Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight

Yiming Mao; Assane Hamidou Abdoulaye; Jiming Song; Xiaowen Yao; Yijie Zhang; Yu Gao; Yiwei Ye; Kai Luo; Wei Xia; Yinhua Chen

doi:10.1371/journal.pone.0282100

. 2023 Apr 20;18(4):e0282100. doi: 10.1371/journal.pone.0282100

Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight

Yiming Mao ^1,^#, Assane Hamidou Abdoulaye ^1,^#, Jiming Song ², Xiaowen Yao ¹, Yijie Zhang ¹, Yu Gao ¹, Yiwei Ye ¹, Kai Luo ¹, Wei Xia ^1,^*, Yinhua Chen ^1,^*

Editor: Vibhav Gautam³

PMCID: PMC10118128 PMID: 37079564

Abstract

The Arabidopsis thaliana ASYMMETRIC LEAVES2 (AS2) gene is responsible for the development of flat, symmetric, and extended leaf laminae and their veins. The AS2 gene belongs to the plant-specific AS2-LIKE/LATERAL ORGAN BOUNDARIES (LOB)-domain (ASL/LBD), which consists of 42 proteins in Arabidopsis with a conserved amino-terminal domain known as the AS2/LOB domain, and a variable carboxyl-terminal region. AS2/LOB domain consists of an amino-terminal (N-terminal) that contains a cysteine repeat (the C-motif), a conserved glycine residue, and a leucine-zipper-like. AS2/LOB domain has been characterised in plants such as A. thaliana, Zea mays, and Oryza sativum. Nevertheless, it remains uncharacterised in cassava (Manihot esculenta). Characterisation and identification of cassava ASL/LBD genes using the computational algorithms, hidden Markov model profiles (PF03195), determined 55 ASL/LBD genes (MeASLBD1 to MeASLBD55). The gene structure and motif composition were conserved in MeASLBDs, while the expression profiles of these genes were highly diverse, implying that they are associated with diverse functions. Weighted gene co-expression network analysis (WGCNA) of target genes and promoter analysis suggest that these MeASLBDs may be involved in hormone and stress responses. Furthermore, the analysis of cis-regulatory elements in promoter regions suggested that MeASLBDs may be involved in the plant phytohormone signal response. The transcriptome data of cassava under biotic and abiotic stresses revealed that MeASLBD46 and MeASLBD47 greatly respond to disease and drought. The MeASLBD47 gene was selected for functional analysis. The result indicated that MeASLBD47 significantly mitigated the virulence of cassava bacterial blight (XamCHN11) through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and Virus-induced gene silencing (VIGS). These findings provided a comprehensive analysis of ASL/LBD genes and laid the groundwork for future research to understand ASL/LBD genes.

Introduction

Organ development in multicellular systems is governed by the activation of a new genetic program and the repression of a previously active program, which is largely governed by epigenetic systems. The prime model for studying such positive and negative control programs is the Arabidopsis thaliana leaf developmental process with an adaxial-abaxial (dorsal-ventral) polarity that is epigenetically regulated by the repressor complex ASYMMETRIC LEAVES1 (AS1)-AS2 [1–4]. AS2 gene has received the most extensive investigation in genetic and molecular research [5]. The AS2 gene in Arabidopsis is a key regulator in the development of flat symmetric leaves with vascular bundles and fine networks of venation systems, the morphology of which appears to have evolved suitably for efficient photosynthesis [5]. For instance, Arabidopsis AS2 regulates leaf adaxial-abaxial partitioning by suppressing the expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3), implying that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation [6]. AS2 specifically binds the CpG-containing sequence in exon 1 of ETT/ARF3, and the binding requires the zinc-finger-like motif in AS2, which is structurally similar to the zinc-finger-CxxC domain in vertebrate DNA methyltransferase1 [6]. In addition, AS2 is involved in epigenetic repression of the abaxial ARF4 and class 1 KNOX homeobox genes by forming a complex with the MYB protein. The cooperative action of various modifier genes significantly boosts the repressed expression of these genes by AS2 [6].

Transcription factor (TF) families play critical roles in plant growth, development, and environmental stress responses [7]. The ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family is an important plant-specific TF family [8]. These members are also named the Lateral Organ Boundary (LOB) Domain (LBD). ASL/LBDs are defined by a highly conserved LOB domain of approximately 100 amino acids (aa) [9]. AS2 gene belongs to the plant-specific AS2/LOB protein family, which contains 42 members in Arabidopsis with a conserved amino-terminal domain known as the AS2/LOB domain, and a variable carboxyl-terminal region; however, little is known about their functions [9–13]. The AS2/LOB domain contains (i) a zinc-finger-like motif (CX₂CX₆CX₃C) required for binding specific DNA sequences (5’ GCGGCG 3’), and the interaction with basic helix-loop-helix (bHLH) proteins could reduce this affinity; (ii) an invariant glycine residue, Gly-Ala-Ser (GAS), critical for the biological function of AS2/LOB proteins in Arabidopsis; and (iii) a leucine-zipper-like (LZL-region; LX₆LX₃LX₆L) which may play a role in the protein-protein interactions to form homo and/or hetero multimers [9–11, 14]. Importantly, the crystal structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) was crystallized and determined [15]. The structure mainly consists of a zinc finger, a GAS motif consisting of two α-helices, a highly conserved five-residue motif (Asp-Pro-Val-Tyr-Gly, known as DPVYG motif), and an amphipathic α-helix with the feature of leucine zipper-like coiled-coil element. According to biochemical, molecular modeling, and small-angle X-ray scattering analysis, dimerization is important for cooperative DNA binding and palindromic DNA discrimination through a molecular calipers mechanism [15]. Based on the presence of the LZL-region, the ASL/LBD gene family can be classified at least into two classes: “Class I” contains all these conserved domain motifs, whereas “Class II” contains only structural motifs similar to the zinc-finger-like motif [9–11, 16]. “Class I” is further divided into two subclasses (Ia and Ib) [5]. A base substitution mutation in the conserved glycine codon of ASL/LBD5 causes the typical phenotypic alterations observed in numerous ASL/LBD mutants, making this residue essential for ASL/LBD function [11, 14]. In addition, the ASL/LBD5 mutant, which contains a mutation in the region encoding the carboxy-terminal (C-terminal) half of ASL/LBD, results in the typical mutant phenotype; thus, the C-terminal half also plays a role in ASL/LBD function [17].

The first ASL/LBD gene (AT5G63090) was identified in A. thaliana [9]. Since then, ASL/LBD genes have been continually reported in various plants and associated with diverse functions (S1 Table in S1 File). For instance, AS2 (AS2/LOB6) in Arabidopsis is involved in a regulatory loop that maintains the shoot meristem and controls leaf polarity and flower development by interacting with an MYB motif encoded by AS1, recently defined as the SANT domain [12, 14, 18]. In addition, it was found that the loss-of-function of AS2/LOB20 in Arabidopsis enhanced resistance to the root-infecting vascular wilt pathogen Fusarium oxysporum [19]. Furthermore, the expression profiles of A. thaliana (At) LBD members indicated that pathogen inoculation induced the expression of AS2/LOB37 and AS2/LOB38 [19]. CsLOB1, a homolog of AS2/LOB1 and AS2/LOB11, acts as a target of transcription activator-like (TAL) effectors following infection with bacterial canker disease [20]. These findings imply that the ASL/LBD genes play important roles in plant defence responses.

Cassava (2n = 36; Manihot esculenta Crantz) is a major staple crop in tropical regions and the third most consumed grain after rice and maize [21–23]. M. esculenta Crantz originated from its wild ancestor, M. esculenta ssp. Flabellifolia [24]. The cassava root crop provides staple food for over 700 million people worldwide [23, 25]. Cassava is highly drought-resistant, and its storage roots can be preserved in the soil for a few years, making it an essential carbohydrate source to alleviate global famine [26]. Besides, it is an ideal feedstock crop for bioenergy, biomaterials, and animal feeds due to its advantageous agricultural characteristics and high starch quantity and quality [27, 28]. Nonetheless, cassava production is constrained by several plant pathogens that threaten the food security of millions of people worldwide [29–31]. Cassava’s most serious bacterial disease is Cassava Bacterial Blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam) [32]. CBB threatens food security in tropical regions and can generate up to 100 percent losses under favourable climatic conditions (CABI, 2015; FAO, 2008) [33]. The rapid spread of CBB in some cassava-producing regions and the emergence of new disease reports in regions where cassava is a staple crop highlight the necessity of developing novel methods to control this plant disease [30, 34–36]. Cassava genome publication lays the groundwork for genome-wide analysis of new gene resources [37]. Nonetheless, studies on the molecular mechanisms of CBB resistance are scarce [38]. The characteristics underlying the biotic stress response of cassava remain largely unknown. This investigation was conducted to identify the ASL/LBD gene family in cassava and determine LOB expression profiles under pathogen stress. Identifying and characterising the ASL/LBD genes and determining their expression profiles under biotic stress could provide valuable insight for plant disease control.

This study identified 55 M. esculenta ASL/LBDs (MeASLBDs) from the cassava genome. The phylogeny, conserved motifs, gene structures, and expression profiles of MeASLBDs were thoroughly determined. Moreover, potential target genes were identified via promoter analysis, and a co-expression network was constructed along with MeASLBDs based on a transcriptome dataset derived from pathogen treatment. The MeASLBD47 gene expression was highly induced following inoculation with cassava bacterial blight pathogen (XamCHN11) and was selected for functional analysis. The results indicated that MeASLBD47 significantly mitigated the virulence of cassava bacterial blight (XamCHN11) through qRT-PCR and Virus-induced gene silencing (VIGS). These findings provide invaluable insight for further physiological and functional studies of the AS2/LOB domain in cassava and highlight the possible functions in pathogen response.

Materials and methods

Plant material and growth conditions

The cassava cultivar used in this study were SC8, Arg7, W14, and KU50. The stems were cut into three-node sections and grown in a pest-free sterile phytotron at temperatures ranging from 35°C to 25°C under a 16/8-h photoperiod and 80% relative humidity. The leaves (30-day-old) were inoculated with Xanthomonas axonopodis pv. Manihotis (XamCHN11) [32]. The leaves of five cassava plants (nine leaves per plant) were inoculated by dropping 10‐μL of XamCHN11 suspension of 1 × 10⁸ CFU/mL [optical density at 600 nm (OD₆₀₀) = 0.1] into a 2‐mm‐diameter ring. Cassava seedlings were sampled at 1 h, 3 h, 6 h, and 9 h post-inoculation. All samples were frozen in liquid nitrogen and stored at -80°C until use. To quantify the size of the lesion, typical Xam symptoms, such as necrosis and chlorosis around the inoculation point, were considered. At 3 days post‐inoculation (dpi), lesions were measured on eight leaves per treatment and then plotted. The experiments were conducted at least twice with identical results.

RNA isolation, qRT-PCR analysis, and statistics analysis

Total RNA was isolated with the RNAprep Pure Plant Kit (DP441, TIANGEN, Beijing, China). RNA concentration determination, DNase I, RNase-free (1 U/μL) treatment, cDNA synthesis, qRT-PCRs, and data analysis were performed following published protocols with minor modifications [39, 40]. Photometric UV-Vis RNA/DNA quantification was performed using NanoDrop One/One^C. The cDNA was synthesized with the FastKing RT Kit (TIANGEN, Beijing, China). The gene-specific primers were designed with the Primer 3.0 program (S2 Table in S1 File). qRT-PCR was performed in a reaction system of 20 μL containing 10 μL TB Green Premix Ex Taq II, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 2 μL cDNA, and 6 μL ddH2O. The PCR amplification conditions were set as follows: denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 35 s, 95°C for 15 s, and 60°C for 1 min. The EF1α gene was selected to calculate the relative fold differences using the 2^-ΔΔCT method [ΔΔC_T = (Ct_{target gene}—Ct_EF1α)] [40]. Statistics analysis was conducted using SPSS software. Rstudio software was used to infer the heatmap. Three sets of biological replicates and three sets of technical replicates were analysed.

Generation of pCsCMV VIGS construct and agroinfiltration

VIGS tool (https://vigs.solgenomics.net/) was applied to analyse the CDS sequence of the gene and obtain the optimal silencing fragment for the construction of VIGS vector [41, 42]. 487 bp cassava PDS (Manes.05G193700) and 237 bp MeASLBD47 (Manes.12G110600) fragments were amplified using cassava cDNAs as a template and the corresponding primer pairs (S2 Table in S1 File). Subsequently, the amplified fragments were cloned into pCsCMV vector to generate pCsCMV-PDS and pCsCMV-AS2/LOB47 using Nimble Cloning [43]. pCsCMV-NC was utilised as a negative control (NC). The pCsCMV-NC-based constructs were transformed into Agrobacterium tumefaciens (strain GV3101). A single colony of A. tumefaciens (GV3101) for each strain was incubated for 20–36 h at 28°C in Luria–Bertani (LB) medium (10 mL) containing 25 mg/mL kanamycin and 25 mg/mL rifampicin. The bacterial cultures were then centrifuged at 4,000g for 5 min and were resuspended in agroinfiltration buffer [10 mM MgCl₂, 10 mM 2-(N-Morpholino) ethanesulfonic acid, and 100 μM acetosyringone] for reaching an optical density of 1.0 at 600 nm (OD₆₀₀) [44]. The agrobacterium suspension was kept at room temperature for 3 h in the dark before inoculation. 2-week old cassava plants were used for agroinfiltration using a 1-mL needleless syringe. The back sides of the healthy and fully developed leaves from the middle of each plant were selected for agroinfiltration. Inoculations were administered at 8–10 spots on both sides of the main vein per leaf to increase the infiltrated leaf area.

Identification and sequence analysis of MeASLBD genes

The cassava genome sequence was downloaded from Phytozome (http://www.phytozome.net) [45]. The AS2/LOB protein sequences of A. thaliana were retrieved from TAIR 12.0 (www.arabidopsis.org) [46]. The hidden Markov model (HMM) -based profile (Pfam: PF03195) of the putative AS2/LOB domain was built using the HMMER program (http://hmmer.org/) [47, 48]. AS2/LOB domain obtained from the Pfam database (http://pfam.xfam.org/) [49] was used as a query to identify all potential AS2/LOB protein sequences using a BLASTp search E-value threshold of 1.0 × 10⁻¹⁰. The putative AS2/LOB domain of the candidate sequences was further confirmed by the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) and the SMART database (http://smart.embl-heidelberg.de/). The MW and pI of cassava AS2/LOB proteins (MeASLBDs) were calculated with the online program ExPASy (https://www.expasy.org/) [50].

Phylogenetic tree construction, conserved motifs, and structure analysis

To determine the evolutionary relationships between M. esculenta and A. thaliana ASL/LBD gene family, phylogenetic analysis was performed with MEGA 7.0 by constructing a minimum evolution (ME) phylogenetic tree using substitution models G + I with 1,000 bootstrap replicates to support statistical reliability [51]. The full-length amino acid sequences of M. esculenta and A. thaliana AS2/LOB protein sequences were aligned using the ClustalX program with the default parameters. The results were displayed with DNAMAN 9.0 (Lynnon Biosoft) software. The Multiple Em for Motif Elucidation (MEME, version 5.4.1.) tool (http://meme-suite.org/tools/meme) was used to predict the conserved motifs of the MeASLBD protein sequences [52]. The relative parameters were set to an optimum motif width of 25–50 and a maximum number of 10 motifs. The exon-intron structure of MeASLBD genes was analysed using the Gene Structure Display Server (http://gsds.gao-lab.org/) [53].

Chromosomal location, gene duplication event, gene collinearity, and synteny analysis

The chromosomal location information of 55 MeASLBD genes was extracted from the cassava genomic annotation file GFF3 (general feature format), retrieved from Ensembl Plants (http://plants.ensembl.org/index.html). The chromosomal distribution of MeASLBD genes was visualised using TBtools software [54]. Duplication detection for MeASLBD genes was performed using the Multiple Collinearity Scan Toolkit (MCScanX) with an E-value of 10⁻⁵ [55]. The homologous regions of the MeASLBD genes were identified with the MCScanX (http://chibba.pgml.uga.edu/mcscan2/) (https://github.com/wyp1125/MCScanX) program to determine the level of synteny [55]. Tandem and segmental duplication events were identified and visualised with Circos software (http://circos.ca/) version 0.69 [56]. The K_a/K_s value was calculated as the ratio of the number of nonsynonymous substitutions per nonsynonymous site (K_a) to the number of synonymous substitutions per synonymous site (K_s) over a given period.

Cis-acting element analysis and identification of MeASLBDs target gene

The promoter regions of 55 MeASLBD genes, the upstream (2000 bp) of the translation initiation codons, were extracted from the Phytozome database of the cassava genome (https://phytozome.jgi.doe.gov/). These sequences were applied in PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html) [57] to predict the cis-acting elements present in the promoter regions of the MeASLBDs, and visualised with TBtools. The genes containing the motif (5’ GCGGCG 3’) in the upstream promoter region are candidate target genes for MeASLBD. Blast2GO tool was used to annotate the candidate target genes [58].

Gene expression profiles of MeASLBDs in cassava

The expression profiles of 55 MeASLBD genes in root and leaf tissues of cassava cultivars Argentina 7 (Arg7), wild subspecies 14 (W14), and Kasetsart University 50 (KU50) were analysed. RNA-seq data of the Arg7, W14, and KU50 were retrieved from NCBI database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93098) (S7 Table in S1 File). HISAT2 (https://github.com/DaehwanKimLab/hisat2) software [59] was applied with default parameters to map the reads. Under default parameters, the StringTie program [60] was applied to assemble transcripts and compute reads per kilobase million (RPKM) values. The cassava AS2/LOB family heatmaps were established with log2-transformed RPKM values and visualised with Tbtools [54]. The min-max method was used to normalise the RPKM values.

Co-expression analysis of MeASLBDs and identification of MeASLBD interacting genes by WGCNA

A co-expression analysis of MeASLBD and its target genes was generated using 55 selected transcriptomes (S3 Table in S1 File). The RPKM values of MeASLBDs and their predicted target genes were screened, and genes with an RPKM value < 1 were removed. Weighted Gene Co-expression Network Analysis (WGCNA) was performed using the Rstudio environment to identify the selected co-expression module [61]. The selected co-expression module is considered a cluster of highly related genes. The module genes showing expression patterns consistent with MeASLBDs were selected for Cytoscape visual analysis [62].

Results

Identification and chromosome localisation of cassava ASL/LBD genes

55 ASL/LBD genes were identified in cassava and named MeASLBD (MeASLBD1 to MeASLBD55). The predicted protein products of MeASLBDs ranged from 116 to 429 aa, with the molecular weight (MW) varying from 13.14 (MeASLBD28) to 46.78 kDa (MeASLBD55). The isoelectric point (pI) of their proteins varied from 4.67 (MeASLBD38) to 9.02 (MeASLBD16). Through chromosomal localisation analysis, 54 MeASLBD genes were unevenly distributed on the 18 cassava chromosomes (S1 Fig in S1 File). However, MeASLBD19 was localized on an unanchored scaffold004600 (S1 Fig in S1 File). The largest number of MeASLBD genes was found on chromosome 7 (7, 12.7%), followed by chromosomes 12 (6, 10.9%), 5 (5, 9%), 10 (5, 9%), 14 (5, 9%), 6 (4, 7.2%), and 13 (4, 7.2%). The remaining chromosomes contained one or two MeASLBD genes.

Evolutionary relationship and gene structure analysis of the MeASLBD genes

To investigate the evolutionary relationship of the MeASLBD genes, 43 AS2/LOB proteins of A. thaliana (AtASL/LBD) and 55 MeASLBD were used to construct a ME tree (Fig 1) MeASLBDs were classified into two major classes: Class I (46) and Class II (9). Class I ASL/LBD genes contain a conserved CX₂CX₆CX₃C zinc-finger-like motif and an LX₆LX₃LX₆L LZL-region, while Class II ASL/LBD genes contain only a conserved zinc-finger-like motif. Forty-six MeASLBDs (83%) were clustered into Class I. Class I was subdivided into seven subgroups (Class Ia to Class Ig). Class Ia contained the highest number of MeASLBD (13), followed by 11, 11, 6, 2, 2, and 1 MeASLBDs belonging to Class Ib, Ic, Ie, Id, and If, respectively. Nine MeASLBDs belonged to Class II, subdivided into two distinct subgroups: Class IIa (4) and Class IIb (5). Interestingly, this result is consistent with Arabidopsis ASL/LBD genes classification [9]. Previous studies showed that many AtASL/LBDs genes (Class I) were involved in lateral organ development [10]. Class II ASL/LBD genes are related to metabolism [10]. The phylogenetic analysis of A. thaliana and M. esculenta indicated that MeASLBD genes might have similar biological functions as AtASL/LBDs.

Fig 1 — The proteins *AtASL/LBD* and *MeASLBD* sequences were used to construct a minimum evolution (ME) tree. The tree was divided into two classes (Class I and II) and nine subclasses (Ia-g and IIa-b).

Gene structure analysis (Fig 2A) showed that MeASLBD genes have a simple structure with 2 to 4 introns and 1 to 4 exons. The majority (45) of the MeASLBD (82%) genes contains two exons, three MeASLBD genes (5%) contain one exon, three MeASLBD genes (5%) contain four exons and one MeASLBD gene (8%), clustered in Class Ig, contains five exons (Fig 2C). 40 MeASLBD coding regions contained a splicing site in the AS2/LOB domain (Fig 2C). 11 MeASLBD noncoding regions (UTR) contained an intron (Fig 2C). Ten putative motifs were identified using the MEME tool (Fig 2B). The conserved motifs of the MeASLBD protein sequences ranged from 25 to 50 aa (motifs 8 and 1, respectively) in length and contained between one (MeASLBD55) to six (MeASLBD14, MeASLBD15, MeASLBD20 and MeASLBD21) motifs. Motifs 1 and 2 were conserved in all MeASLBD protein sequences except MeASLBD55 which contained only motif 2. Cluster analysis showed that Class Ia, Id, Ig, and II have each similar gene structures. Motifs 3, 5, 6, and 10 were only conserved in MeASLD proteins of Class I, whereas motifs 8 and 4 were only conserved in MeASLDs proteins of Class II, suggesting that they are evolutionary divergent. Notably, motif 2 is conserved in all MeASLBD proteins. The MeASLBD proteins with similar motif compositions and gene lengths indicate they may share similar functions.

Conserved motif analysis of MeASLBD genes

Multiple sequence alignment of the MeASLBD protein sequences using ClustalX showed that all sequences contained a zinc-finger-like motif (CX₂CX₆CX₃C) at the N-terminus (Fig 3). The LZL-region (LX₆LX₃LX₆L) was only found in MeASLBD proteins of Class Ⅰ, and 20 MeASLBD protein sequences (MeASLBD4, MeASLBD8, MeASLBD9, MeASLBD10, MeASLBD11, MeASLBD12, MeASLBD14, MeASLBD15, MeASLBD20, MeASLBD21, MeASLBD22, MeASLBD24, MeASLBD25, MeASLBD26, MeASLBD27, MeASLBD29, MeASLBD31, MeASLBD32, MeASLBD35, MeASLBD45) contained a complete motif, while the remaining MeASLBDs contained an uncomplete motif. Moreover, most MeASLBD genes contained a complete ICG/GAS-region, except for MeASLBD55.

Fig 3 — (a) Multiple sequence alignment of *MeASLBD* conserved domains by DNAMAN. (b) Visualisation of the CX₂CX₆CX₃C motif, ICG/GAS-region motif, and LX₆LX₃LX₆L LZL-region.

Duplication, Collinearity, and synteny analysis

Genomic segmental and tandem duplications represent the two main driving forces of gene family expansion. MCScanX was applied to explore the gene duplication events in the MeASLBD gene family (Fig 4). Seven pairs of tandem duplicated genes were identified on chromosomes 1 (MeASLBD31/27), 5 (MeASLBD26/35), 7 (MeASLBD13/55), 10 (MeASLBD24/6), 12 (MeASLBD20/14), and 13 (MeASLBD21/15). 42 MeASLBD genes were segmentally duplicated, implying segmental duplication was a major driving force in the evolution of the MeASLBD gene family compared to tandem duplication. In addition, the K_a/K_s for 34 ASL/LBD gene pairs were determined (S4 Table in S1 File). The K_a/K_s ratio of ASL/LBD gene pairs varied from 0.0708 to 0.5342. The K_a/K_s ratios were less than 1.0, indicating that these genes might have undergone purifying selection during the evolution process. Collinearity analysis revealed chromosomal duplication, translocation, and inversion in the MeASLBD gene family (Fig 5). Genome Synteny and Collinearity Analysis show that ASL/LBD is relatively conserved in Arabidopsis and cassava. AtLBD has a high degree of height with MeASLBD.

Fig 4 — The black lines show the position distribution of *MeASLBD* genes on 18 chromosomes. The red lines show the collinear gene pairs of *MeASLBDs*.

Fig 5 — Orange represents the chromosomes (Chr) of *Arabidopsis thaliana*, Green represents the chr of cassava, Red lines represent genes containing Homologous. Pt represents the Chloroplast, Mt represents mitochondrion. The collinearity analysis was conducted using MCScanX and visualized with TBtools.

Analysis of putative cis-elements of the MeASLBD gene

Analysis of cis-regulatory elements in promoter regions helps understand the mode of gene regulation and predicts the functions of genes. The online tool Plant CARE was used to investigate the cis-elements of the MeASLBD genes within a 2000-bp upstream region (Fig 6). Among the seven known elements, the hormone-responsive, environmental stress-related, and development-related elements were the core physiological processes represented by the regulatory elements. The hormone-responsive elements present in the MeASLBD gene family were found to be related to auxin-producing genes (TGA-element, AuxRE, and AuxRR-core), gibberellin (GARE-motif, P-box, and TATC-box), salicylic acid (TCA-element), abscisic acid (ABRE), methyl jasmonic acid (CGTCA-motif, TGACG-motif), and ethylene (ERE), the elements related to ethylene, jasmonic methyl acid, and abscisic acid were abundant, indicating that these genes may respond to phytohormone signals and/or abiotic stresses. Environmental stress-related elements, including anaerobic induction, defence and stress responsiveness (TC-rich repeats), low-temperature responsiveness, and enhancer-like elements, are involved in anoxic-specific inducibility (GC motif). In addition, meristem expression was found in development-related elements, suggesting that these genes might be involved in the development of plant meristems.

Fig 6 — Tbtools software was used to search the cassava promoter elements, count the number of promoters with the same function, and used TBtools for visualization. The color bar represents the number of the putative cis-elements of the *MeASLBD* gene ranging from light green to dark green.

Expression analysis of MeASLBD genes in different tissues and organs

The expression profiles of MeASLBD genes in different tissues (leaves and roots) of the cassava cultivars Arg7, W14, and KU50 were analysed (Fig 7). In cultivars Arg7, KU50, and W14; 1% (5/55), 9% (16/55), and 47% (26/55), respectively, of the MeASLBD genes were expressed in leaves and roots. MeASLBD1, MeASLBD2, MeASLBD11, MeASLBD46, MeASLBD47 and MeASLBD51 were highly expressed (RPKM value > 10) in roots and leaves, while 26 MeASLBDs had low or no expression in the three cassava cultivars.

Several genes displayed differential expression profiles between tissues or different cultivars. MeASLBD1 and MeASLBD2 were highly expressed in the roots of W14 but at low levels in the leaves. MeASLBD47 and MeASLBD51 displayed higher expression in W14_root and KU50_root_75d. In addition, MeASLBD47 was also expressed in KU50_leaf. These results suggest the genotype-dependent tissue expression of these genes.

Expression profiles of the MeASLBD genes in response to drought and XamCHN11 infection

The expression profiles of the 55 MeASLBDs in root and leaf tissues of two cassava cultivars (Arg7 and W14) under drought stress were investigated (Fig 8). The MeASLBD gene was expressed higher in roots than in leaves for both cultivars. Six MeASLBDs (MeASLBD7, MeASLBD10, MeASLBD46, MeASLBD47, MeASLBD51 and MeASLBD52) had significant differential expression under drought stress. The MeASLBD51 expression was significantly induced in Arg7, but decreased in W14 for the root tissues. Meanwhile, the expressions of MeASLBD10, MeASLBD46, and MeASLBD47 were significantly altered in both roots and leaves. MeASLBD7 and MeASLBD52 were highly expressed in Arg7_CK12 root but were significantly downregulated under drought stress.

Fig 8 — The ratio of the experimental group to the mock group was log2 transformed. W14_ck12l, W14D12, Arg7_ck12, and W14_CK12 represent leaves. And 14_ck12, W14D12 root, Arg7_ck12 root, and W14_CK12 root stands for root (S7 Table in S1 File). D stands for drought. (https://www.ncbi.nlm.nih.gov/biosample?Db=biosample&DbFrom=bioproject&Cmd=Link&LinkName=bioproject_biosample&LinkReadableName=BioSample&ordinalpos=1&IdsFromResult=246428).

Besides the drought stress, the expressions of some MeASLBDs were also influenced under XamCHN11 stress, including MeASLBD10, MeASLBD12, MeASLBD46, and MeASLBD47. Among these four genes, the MeASLBD10 gene expression was steadily downregulated over infection time, whereas the MeASLBD46 and MeASLBD47 gene expressions were upregulated. Analyses of the transcriptome data of the cassava cultivars (Arg7 and W14) under stress treatments showed that some ASL/LBD genes respond significantly to abiotic and biotic stresses.

Validation of six MeASLBD genes in response to XamCHN11 via qRT-PCR

Six MeASLBD genes were randomly selected for qRT-PCR validation under a pathogenic bacterium (Xam) stress. The results of qRT-PCR showed that the MeASLBD genes (MeASLBD1, MeASLBD2, MeASLBD12, MeASLBD13, MeASLBD46, and MeASLBD47) had a different expression level at 1 h, 3 h, 6 h, and 9 h post-inoculation (Fig 9). The expression of MeASLBD13 was upregulated at 6 h and 9 h post-inoculation but was downregulated at 1 h and 3 h post-inoculation. MeASLBD46 was significantly upregulated at 1 h, 3 h, 6 h, and 9 h post-inoculation. MeASLBD47 was upregulated progressively after inoculation but decreased slightly at 9 h post-inoculation. The expression levels of MeASLBD1, MeASLBD2, and MeASLBD12 increased significantly at 6 h post-inoculation and then decreased at 9 h post-inoculation. These gene expression levels were consistent with the transcriptomic data. The results indicated that the most significant response of MeASLBD genes to pathogens occurred at 6 h post-inoculation. MeASLBD46, MeASLBD13, and MeASLBD47 expression levels were significantly expressed following treatment with XamCHN11.

Fig 9 — Accumulation levels of *MeASLBD* transcripts were determined by qRT-PCR (n = 3). EF1α was the internal control. Transcription levels were analysed using the comparative “2^−ΔΔCt” method. Symbols represent the mean ± SD. Groups designated by the same letter are not significantly different, while those with different letters (“*” or “**”). “**” (p < 0.01) and “*” (0.01 < p < 0.05).

Identification of candidate genes coexpressed with MeASLBD genes

One thousand four hundred forty genes contain a 5’ GCGGCG 3’ motif in the promoter of the cassava genome, a putative motif bound by ASL/LBD genes. GO enrichment analysis revealed that these genes are mainly involved in metabolic, cellular, and biological processes. These genes are mainly allocated to cellular components for cells, cell parts, and organelles. Binding and catalytic activity are the main categories under molecular functions (Fig 10).

Fig 10 — Red is biological processes, green is cell composition, and blue is molecular functions.

To further analyse the relationship between MeASLBD genes and the predicted downstream target genes, a set of 55 transcriptomes derived from XamCHN11 infected cassava leaves was utilised to perform WGCNA co-expression analysis. The RPKM values of 55 MeASLBD genes and 1,450 target genes were determined. 347 genes with the expression coefficient of variation (CV) >1 and RPKM >1 were retained for WGCNA (S5 Table in S1 File). Highly interconnected gene sets known as modules were obtained via WGNCA analysis. The 347 genes could be divided into four modules via WGCNA, namely blue (67), brown (37), turquoise (217), and grey (25) (S6 Table in S1 File). Grey was considered irrelevant to the sample; therefore, this module was removed. The gene co-expression modules for the MeASLBD and target genes are shown in Fig 11. The enrichment analysis showed that 16 genes were involved in plant-pathogen interaction, 6 in the MAPK signalling pathway, and 14 in defence.

Fig 11 — The genes in the WGCNA module showed expression patterns and consistent with those of *MeASLBDs*.

Silencing of MeASLBD47 reduced the lesion area and enabled differential expression of target genes

VIGS approach was applied to silence MeASLBD47 gene. The silencing efficiency was examined using qRT-PCR. The results showed that the abundance of MeASLBD47 in cassava species was reduced compared with NC (Negative Control), indicating that MeASLBD47 was efficiently silenced (Fig 12A). Cassava was inoculated with pCsCMV-AS2/LOB47, pCsCMV-NC and 10 mM MgCl2 (Mock) for 3 days (Fig 12B). After three days, lesions appeared on the NC leaves, whereas pCsCMV-AS2/LOB47 showed no obvious lesions. The result suggests that MeASLBD47 silencing enhances plant resistance by reducing lesion areas. Through the co-expression network constructed using WGCNA, the possible downstream genes regulated by MeASLBD genes were predicted. The genes expressed in the regulatory network were detected based on VIGS (Fig 12C). Among them, the expressions of HSFC1 and GAD1 were significantly downregulated, whereas HSP90 was significantly upregulated, indicating that they were affected by the effect of VIGS. After three days, TFIIA-S expression was slightly affected. Overall, MeASLBD47 might affect plant resistance by regulating HSFC1 and GAD1.

Fig 12 — (a) Validation of the silencing efficiency of *MeASLBD* by qRT-PCR, pCsCMV-NC represents an empty vector. (b) The phenotypes of the cassava leaf infected with *Xam*CHN11 following inoculation with pCsCMV-*AS2/LOB*47 or empty vector (pCsCMV-NC), 10 mM MgCl2 (mock for *Xam*CHN11). The lesion area of cassava leaves was calculated using the ImageJ tool at 200 pixels/cm. (c) Differential expression of target genes in the WGCNA network upon silencing of *MeASLBD47*. “*”: significantly different.

Discussion

Cassava (M. esculenta. Crantz) is a starch-rich, woody tuberous root crop that is important for food and as a potential bioenergy crop. Despite its importance in food and bioenergy, cassava cultivation is fraught with difficulties (abiotic and biotic stresses), resulting in decreased crop yield. Most are susceptible to pests and diseases threatening existing cultivars [63, 64]. AS2/LOB genes are widely present in plants and are important plant-specific process regulators [13]. To date, several ASL/LBD genes have been identified in A. thaliana (43) [9], Eucalyptus grandis (46) [65], Glycine max (90) [66], and other species. However, systematic studies of MeASLBD genes have not been reported so far. The availability of whole-genome sequences of M. esculenta helps in the genome-wide characterisation of AS2/LOB genes, which may be used to improve crop yield in the field. In this study, 55 MeASLBD genes were identified, indicating that the AS2/LBD gene family has largely retained a fixed function in the genetic evolution of different species. Two types of terminology were listed side by side (ASLs and LBDs). Such terminology might provide an advantage in discussing the evolutionary developmental biology of the AS2/LOB protein family [5]. The AS2/LOB gene family in M. esculenta is similar to the estimates for other reported plant species. The MeASLBD genes were divided into two classes (I and II) and nine subclasses (Ia~Ig, IIa, and IIb) based on the structure of the LOB domain (Fig 1). Forty-six MeASLBD genes (83%) belonged to Class I, and nine MeASLBD genes (17%) belonged to Class II. Previous studies also reported that 84% and 16% of the ASL/LBD genes in A. thaliana belonged to Classes I and II, respectively [11]. 98 ASL/LBD genes from A. thaliana and M. esculenta were further classified into nine subclasses (Ia-If and IIa-IIb) based on their phylogenetic relationships (Fig 1), and were consistent with those previously reported [67]. Homologous genes in the same class or subclass might share the same function. For instance, ASL15/LBD17 was proposed to play a similar role to ASL18/LBD16, ASL16/LBD29, ASL24/LBD33, and ASL20/LBD18 involved in auxin-induced lateral root formation, all of which belong to the close narrow clades of the tree [68–71]. These results imply that our finding of homologous genes may share similar functions.

The origin and evolutionary history of the ASL/LBD proteins remain largely unexplored. ASL/LBD proteins are streptophyte-specific proteins and likely evolved from the charophycean green algae [72]. Few TF families evolved within land plants, implying the origin of a new TF family is not critical for land plant evolution. Nevertheless, the increased diversity of several TF families, such as ASL/LBD, suggests that they may have been instrumental in terrestrialization [73]. Paleobotanical studies indicate that roots evolved at least twice independently during the evolution of land plants, once in lycophytes and once in euphyllophytes. Auxin promotes the initiation of postembryonic roots in both groups but from different cell types. ASL/LBD proteins act directly downstream of auxin in several euphyllophytes, including Arabidopsis, maize, and rice, and are conserved elements required for root initiation [72]. However, a clade with no lycophyte sequence specifically associated with root development was inferred, implying that root initiation in lycophytes and euphyllophytes is mediated by distinct genetic mechanisms downstream of auxin [17]. In contrast, leaves evolved independently in lycophytes and euphyllophytes [74]. The MeALSBDs evolutionary relationships provide an understanding and could contribute to determining the origin of the ASL/LBD protein family.

Structural analysis effectively extracts valuable information about gene duplication events and phylogenetic relationships within gene families. Gene duplication events, followed by neofunctionalisation, were enough to drive molecular diversification [73, 75]. MeASLBD genes have simple gene structures (Fig 2B). Most MeASLBD members within the same subgroup showed different exon/intron structure and gene length (Fig 2C), slightly different from A. thaliana ASL/LBD genes [9]. Therefore, ASL/LBD gene structures might not be relatively conserved in different species. In addition, cassava contained 55 ASL/LBD genes, 12 more than Arabidopsis and 35 fewer than soybean [60, 66]. Therefore, ASL/LBD genes vary from one species to another, consistent with previously reported ASL/LBD genes [9]. The K_a/K_s ratio indicated that purifying selection had affected the cassava AS2/LOB domain (S4 Table in S1 File). In Arabidopsis ASL/LBD protein sequences comparison of the cloned cDNAs with those of the corresponding genes revealed that these genes were divided into five subtypes based on the positions of the introns in the coding regions (subtypes A to E). 40 MeASLBD genes contained splicing sites in their coding regions (Fig 2C). In comparison, 29 Arabidopsis ASL/LBD genes contained introns in their coding regions.

The ASL/LBD gene family is an important TF family in plant species. Therefore, the function of MeASLBD genes was investigated. Through promoter analysis, most of the MeASLBD promoter positions consisted of ABA, MeJA, and GA response cis-elements (Fig 6), which indicated that MeASLBDs might be involved in the plant phytohormone signal response. In A. thaliana, AtASL/LBD20 is thought to play a role in the JA response [76], and AtASL/LBD40 is downregulated under gibberellin treatment [77]. In cotton, GhLBD123 was significantly upregulated under MeJA treatment [78]. These findings indicate that MeASLBD genes may be involved in hormone and stress responses.

The expression profiles of MeASLBD genes in different tissues (Fig 7) revealed that MeASLBD genes are mainly expressed in the roots, such as MeASLBD2, MeASLBD47, and MeASLBD51, indicating that they might be involved in cassava root development. The transcriptome expression profile of cassava under drought and disease stress was used to study the expression profiles of MeASLBDs (Fig 8). The results revealed that MeASLBD46 and MeASLBD47 responded to drought stress and XamCHN11 infection. MeASLBD46 and MeASLBD47 showed a strong response through qRT-PCR, indicating that they are associated with Xam infection response (Fig 9). The genes MeASLBD46, MeASLBD47, AtASL/LBD37, and AtASL/LBD38 are homologous. MeASLBD46 and MeASLBD47 may thus be involved in nitrogen metabolism, anthocyanin synthesis [79], and stress response.

AS2 can form complexes with other proteins to control different aspects of plant growth and development [1, 11, 80, 81]. AS2 physically associates with AS1 to form a repressor complex that regulates leaf polarity and morphology, inflorescence architecture and fertility, and shoot apical meristem differentiation [1, 11, 80, 81]. AtASL2/LBD10 and AtLBD28 are classified in class I in Arabidopsis [82]. In this study, AtLBD28 was classified in class Id (Fig 1) alongside MeASLBD28 and MeALSBD23. AtLBD28 may influence leaf polarity and morphology, as well as shoot apical meristem differentiation [83]. Therefore, AtLBD28, MeASLBD28, and MeALSBD23 may share similar functions. Moreover, AtASL2/LBD10 participates in both microspore polarization before the first asymmetric division, and germ cell mitosis [84] AtASL2/LBD10 mutants had a 12.7% aborted pollen grain yield, indicating that AtASL2/LBD10 is important for Arabidopsis pollen development [85]. AtASL2/LBD10 was classified in Class Ib (Fig 1), indicating that members such as MeASLBD4 may share similar functions. In addition to their roles in plant development, some Arabidopsis ASL/LBD members play important roles in response to fungal pathogens (e.g., Fusarium wilt) and soil nematodes (e.g., Meloidogyne incognita) [76, 86]. For instance, AtLBD20, primarily expressed in the roots, is a negative regulator of Fusarium wilt resistance and a subset of jasmonate (JA) responses [76]. In this study, AtLBD20, MeASLBD29, and MeASLBD41 were subgrouped into Class Ic (Fig 1), indicating that they may share the same function.

WGCNA is an effective tool for investigating the relationships between different gene sets (modules) [61]. Gene expression modules based on specific species have been efficiently used in diverse varieties. One thousand four hundred fifty genes were identified through promoter analysis. GO enrichment analysis (Fig 11) showed that these genes participate in different regulatory networks. Therefore, AS2/LOB proteins may regulate lateral root growth and plant signal transduction, metabolic processes, and stress responses. MeASLBD46 and MeASLBD47 responded to XamCHN11 infection. In addition, fourteen genes were identified as target genes for MeASLBD regulation via the WGCNA co-expression network (Fig 11). In Arabidopsis, over 350 AtLBD29 target genes have been identified to participate in the regulation of cell reprogramming during callus formation [13, 87, 88]. Some MeASLBD target genes are related to the plant pathogenic bacteria, such as HSP90 [89], TFIIA [90], and GAD1 [91].

VIGS tool was used to silence MeASLBD47 gene and inoculate cassava with XamCHN11 [42]. After 3 days of disease treatment, the water spots on leaves were reduced, and lesions developed slower than NC (Fig 12B). This indicates that the silencing of MeASLBD47 enhances plant resistance against XamCHN11. Simultaneously, qRT-PCR validation was performed on the co-expression network genes (Fig 12A). The result showed that GAD1 and HSFC1 expression was inhibited and, on the contrary, enhanced the expression of HSP90 (Fig 12C). MeASLBD47 might regulate downstream target genes GAD1, HSFC1, and HSP90 to enhance plant resistance. In addition, transcriptome analysis of AS2/LOB37 mutant material in Arabidopsis [79] found that LRR (At1g66090) was significantly upregulated when AS2/LOB37 was deleted. Therefore, the LRR gene in cassava was detected, and the results were consistent with Arabidopsis (S2 Fig in S1 File). These findings provide useful clues for further investigations into gene regulatory networks involved in cassava disease resistance.

Conclusions

Fifty-five ASL/LBD genes were identified from M. esculenta and distributed unevenly among the chromosomes. The MeASLBD gene family is classified into two categories based on gene structure and phylogeny: Class I (46) and Class II (9). Collinearity analysis and the Ks/Ks values indicated that purifying selection was the main force driving the evolution of the MeASLBD gene family. According to the transcriptome data of cassava under biological and abiotic stress, MeASLBD46 and MeASLBD47 were found to have a strong disease and drought response. To better understand the regulatory function of MeASLBDs, the target genes of MeASLBDs were screened and revealed the disease-related genes of HSP90, TFIIA-S, and GAD1. The latter result indicates that MeASLBD46 and MeASLBD47 may participate in the plant response to stress by regulating these target genes. Furthermore, these findings provide valuable information for subsequent elucidation of the role of the ASL/LBD genes.

Supporting information

S1 File. Contains all the supporting tables and figures.

(DOCX)

Click here for additional data file.^{(739.3KB, docx)}

Acknowledgments

The authors thank AJE (https://www.aje.cn) for providing linguistic assistance during the preparation of this manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by National Key Research and Development Program (2018YFD1000500), the Natural Science Foundation of Hainan Province (320QN193) and China Agriculture Research System (CARS-11-hncyh). National Key Research and Development Program (2018YFD1000500) Funding had role in study design and material collection; the Natural Science Foundation of Hainan Province (320QN193) had role in the research of qPCR data collection and analysis; China Agriculture Research System (CARS-11-hncyh) founders had role in RNA-Seq data collection and analysis, manuscript preparation.

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PLoS One. doi: 10.1371/journal.pone.0282100.r001

Decision Letter 0

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27 Apr 2022

PONE-D-22-03287Genome-wide characterisation of cassava LATERAL ORGAN BOUNDARIES domain genes revealed LBD47 involved in bacterial blight defensePLOS ONE

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Reviewer #1: The authors reported the AS2/LOB gene family in cassava (Manihot esculenta), in which members contain plant specific AS2/LOB domain. They identified and characterized 55 ASL/LBD genes (MeLBD1 to MeLBD55) from cassava genome using hidden Markov model profiles. They showed that the expression profiles of these genes were highly diverse. Weighted gene co-expression network analysis (WGCNA) of target genes and promoter analysis suggest that some members of cassava MeLBDs may be involved in hormone and stress responses. Furthermore, they described that the promoter analysis suggested that cassava MeLBDs may be involved in the plant phytohormone signal response.

Major points:

(1) Genome-wide characterisation of the AS2/LOB gene family should be very useful for understanding the function of these genes in cassava. To refine these analysis, they should refer to four papers as following on this subject.

Bowman et al., 2017. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell 171, 287-304 doi.org/10.1016/j.cell.2017.09.030

Machida Y., Suzuki T., Sasabe M., Iwakawa H., Kojima S., Machida C. Arabidopsis ASYMMETRIC LEAVES2 (AS2): roles in plant morphogenesis, cell division, and pathogenesis J. Plant Research 2022 135:3–14 (doi.org/10.1007/s10265-021-01349-6）

Coudert, Y.; Dievart, A.; Droc, G.; Gantet, P. ASL/LBD phylogeny suggests that genetic mechanisms of root initiation downstream of auxin are distinct in lycophytes and euphyllophytes. Mol. Biol. Evol. 2013, 30, 569–572.

Matsumura Y, Iwakawa H, Machida Y, Machida C (2009) Characterization of genes in the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) family in Arabidopsis thaliana, and functional and molecular comparisons between AS2 and other family members. Plant J 58:525–537

According to these papers, LATERAL ORGAN BOUNDARIES domain genes should be written ASYMMETRIC LEAVES2-like/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) genes, MeLBD should be MeASLBD and the LBD gene family should be the AS2/LOB gene family.

In Phylogenetic trees of figure 1, all AtLBD should be written AtASL/LBD, for example, AtASL1/LBD36, AtASL2/LBD10, AtASL3/LBD25, AtASL4/LOB, AtAS2/LBD6, which are classified into Class Ib in this paper. If so, it should be very useful to understand the evolutionary relationship between the ASL/LBD proteins of cassava and those of arabidopsis. In addition, it should be useful for the functionary relationship between them, because the authors can recognize that those with similar numbers are functionally close to each other in arabidopsis.

In figure 1, MeLBD should be MeASLBD, for example, MtASLBD1, MtASLBD2, MtASLBD3 like Marchantia (Bowman et al., 2017).

(2) AS shown in figure 2, it is very interesting that members of the MeASLBD gene family have one to four exons. Class Ia contain one putative splicing site in the AS2/LOB domain at the same site as that in arabidopsis (see Figure 1b in Matsumura et al., 2009) . In arabidopsis a comparison of the sequences of the cloned cDNAs with those of the corresponding genes revealed that 29 genes contained introns in their coding regions, and these genes could be divided into five subtypes on the basis of the positions of the introns in the coding regions (subtypes A–E). The subtype in arabidopsis seems to be corresponded to subtypes in cassaba. They should describe the relationship of exons/introns in their AS2/LOB domain regions between cassaba and arabidopsis.

(3) As shown in Figure 3, multiple sequence alignment of the MeAS2/LOB domain sequences using DNAMAN showed that all sequences contained three subdomeins named C Block, GAS block and L-rich Block. According to the recent two papers as follows, the authors should change the name of subdomain in the AS2/LOB domain. C Block should be written ZF-motif, GAS block should be ICG/GAS-region, L-rich Block should be LZL-region

Machida et al., (2022) J. Plant Research 135:3–14 (doi.org/10.1007/s10265-021-01349-6）

Chen WF, Wei XB, Rety S, Huang LY, Liu NN, Dou SX, Xi XG (2019) Structural analysis reveals a “molecular calipers” mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat. J Biol Chem 294:142–156

(4) The authors described that potential target genes were identified via promoter analysis, and a co-expression network was constructed along with MeLBDs based on a transcriptome dataset derived from pathogen treatment. They found that the MeLBD47 gene expression was highly induced following inoculation with cassava bacterial blight pathogen (XamCHN11), selected for functional analysis. And then the VIGS technique was applied to determine the function of the MeLBD47 gene following XamCHN11 treatment.

They described that one thousand four hundred forty genes contain a 5’ GCGGCG 3’ motif in the only promoter of the cassava genome, a putative motif bound by ASL/LBD genes. Although some members of the AS2/LOB family have been shown interaction to 5’ GCGGCG 3’ motif (Husbands et al., 2007; Vial-Pradel et al., 2018; Chen et al., 2018), function of interaction to 5’ GCGGCG 3’ motif is not known yet. Vial-Pradel et al. found that AS2 binds specifically the sequence containing 5’ GCGGCG 3’ motif in exon 1 of ETT/ARF3 (direct target of AS2), and that the binding requires the ZF-motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1. In addition, Vial-Pradel et al. showed that AS2 did not bind to DNA sequence containing 5’ GCGGCG 3’ like motif of promoter region of ETT/ARF3.

The authors may examine binding to DNA sequence containing 5’ GCGGCG 3’ motif in candidate of target of MeASLBD47 and/or discuss about function of binding to DNA sequence containing 5’ GCGGCG 3’ motif.

Vial-Pradel S., Keta S., Nomoto M., Luo L., Takahashi H., Suzuki M., Yokoyama Y., Sasabe M., Kojima S., Tada Y., Machida Y., Machida C. Arabidopsis zinc-finger-like protein ASYMMETRIC LEAVES2 (AS2) and two nucleolar proteins maintain gene body DNA methylation in the leaf polarity gene ETTIN (ARF3). Plant Cell Physiol. 59(7):1385-1397 (2018).

(5) In Figure 11b, the authors showed that the leaves of pCsCMV-LBD47 plants were inoculated with XamCHN11, and pCsCMV-LBD47 showed no obvious lesions after three days, although lesions appeared in the pCsCMV-NC (control). They should show quantitative data.

(6) Line 349, The authors wrote “Among them, the expressions of HSFC1 HSP90 and GAD1 were significantly downregulated, indicating that they were affected by the effect of VIGS.” However, HSP90 was not downregulated, but upregulated.

(7) There is no Figure legends. The authors should write Figure legends for Fig 1-11.

(8) In References, The authors did not write name of journal. They should write name of Journal of each reference.

(9) Arabidopsis Thaliana should be Arabidopsis thaliana.

Reviewer #2: This paper “Genome-wide characterisation of cassava LATERAL ORGAN BOUNDARIES domain genes revealed LBD47 involved in bacterial blight defense” investigates the LBD genes (MeLBD1 to MeLBD55) in cassava genome using hidden Markov model profiles. A lot of papers have been published on cassava plants infected with Xanthomonas axonopodis. The work showed methodological lacks where necessary, unnecessary statements, and weak discussion. The experimental design data has been poorly explored without any statistical analysis and result description. Moreover, the manuscript has a lot of language and editing issues, which should be corrected and needs extensive revision. In some lines, the inappropriate use of English language makes the manuscript really hard to follow.

Title:

The inappropriate use of English language makes difficult to follow the title. It should be precise and understandable.

Abstract:

Abstract is not well written, there is no proper background, methods, directly results are discussed without any conclusion. There are lots of English language issues that makes the abstract really hard to follow. Some very basic scientific writing errors are there, such as on line 23 “Arabidopsis Thaliana” is written. It should be written as Arabidopsis thaliana.

Keywords:

Authors should use full name instead of abbreviation in this part

Introduction:

The Introduction is poorly written and has many language issues, which need to be corrected. It is advised to get help of English editing service. The literature discussed in one sentence is mostly disconnected from the next sentence, hampering the reading fluency e.g. from Line 43 to 52, 55 to 57, 66 to 67, etc. At some places the meaning of sentence is ambiguous. The information provided need improvement by adding literature relevant to the study conducted. The study objectives are lacking. Authors must clearly present the hypothesis of their study.

Methods:

The methods description also needs improvements. The authors should use scientific language to write protocols and procedures. More clarity is needed in this section like:

• How the Xanthomonas axonopodis infection was given and at which CFU?

• How symptoms were monitored post-inoculation and evaluated according to a severity scale?

• How the HMM profile was created?

• How authors analyzed the conserved domains?

• Which model authors used to construct the phylogenetic tree?

• What was the optimum width of each motif?

• From where authors retrieve the files for analyzing exon-intron structure?

• Authors should express the syntenic relationship of the gene pairs and their respective loci in cassava genome.

• From where authors retrieve GFF3 file for chromosomal location?

• Microcollinearity is valuable to understand the investigation of gene loss during evolution or the evolution of specific genes in a local region. Therefore, authors must do TGT (Triticeae-Gene Tribe) analysis to trace the origin history of the target gene, and also analyze gene pair.

Results:

Result part need substantial improved. The inappropriate use of English language makes the results hard to follow. For example, from lines 178 to 180, etc. Results are ambiguous. The authors must double check the information given in the manuscript as the methodology section saying something different than what is explained in results section including figures.

• The information given in Table 1 is already available on “iTAK - Plant Transcription factor & Protein Kinase Identifier and Classifier”. What new authors have done?

• Figures are not clear. Authors must add high resolution figures to make them understandable. Authors must double check whether the information in the figures are same as they have been written in the results section.

• There are no figure legends. Authors are advised to add them.

• Were the LBD genes groups evenly distributed among chromosomes or different homologous groups?

• Methodology and results of synteny analysis are missing while there is heading in the results.

• Authors must analyze the duplication event

• The results of Exon–intron structure divergences are missing. Authors have only discussed exons but there is no information regarding the introns in all these genes.

• The authors must correlate the phylogenetic relationship among LBDs with exon–intron structure

• In methodology, authors wrote that “The cassava cultivar used in this study was South China 8 (SC8)” while in the results the authors wrote “The expression profiles of MeLBDs in different tissues (leaves and roots) of the cassava cultivar Argentina 7 (Arg7), wild subspecies (W14) and variety Kasetsart University 50 (KU50) were analysed”. Which information is correct?

• What does the heatmap in Figure 6 shows? There is no methodology for this. Results are different from what is said in the methodology.

• The results described in “Expression analysis of MeLBD genes in different tissues and organs” are difficult to understand as the figure which is cited in this don’t show the similar information.

• There is no methodology for drought. How it appear in the results section?

• The transcriptome analysis was done with which treatments and on which plants?

• When silencing of MeLBD47 gene was done? What was its methodology?

• How the statistical analysis was performed?

• What does the stars on the figure 8 show?

Discussion:

Discussion is very poorly written. This section needs substantial improvement in terms of English language and relevant review of literature. In its current form, it is written much more like introduction and results. In text citation also need revision.

Overall, the manuscript seems to be the first rough draft which has to be tidied up in many places and this manuscript in its current form cannot be considered for publication. The manuscript needs extensive revisions before considering it for publication.

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Reviewer #1: No

Reviewer #2: Yes: Hamid Manzoor

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PLoS One. 2023 Apr 20;18(4):e0282100. doi: 10.1371/journal.pone.0282100.r002

Author response to Decision Letter 0

15 Jul 2022

Journal Requirements:

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Response: Thanks for providing us with the PLOS ONE style templates. The manuscript has been thoroughly improved following PLOS ONE's style requirements.

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"This study was supported by the Natural Science Foundation of Hainan Province (320QN193) and the China Agriculture Research System (CARS-11)."

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Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Response: This study was supported by National Key Research and Development Program (2018YFD1000500), the Natural Science Foundation of Hainan Province (320QN193) and China Agriculture Research System (CARS-11-hncyh).

National Key Research and Development Program (2018YFD1000500) Funding had role in study design and material collection; the Natural Science Foundation of Hainan Province (320QN193) had role in the research of qPCR data collection and analysis; China Agriculture Research System (CARS-11-hncyh) founders had role in RNA-Seq data collection and analysis, manuscript preparation.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This study was financially supported by National Key Research and Development Program (2018YFD1000500) and China Agriculture Research System (CARS-11-hncyh)."

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"This study was supported by the Natural Science Foundation of Hainan Province (320QN193) and the China Agriculture Research System (CARS-11)."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Thanks for pointing it out. We have removed the funding from the manuscript.

Upon re-submitting your revised manuscript, please upload your study's minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

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Response: We had uploaded the latest version of the clean manuscript as the main article file.

8. Please amend the title either on the online submission form or in your manuscript so that they are identical.

Response: Sorry for the mistake. We had revised the online submission form. The tile of the form and the manuscript were identical.

Attachment

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PLoS One. doi: 10.1371/journal.pone.0282100.r003

Decision Letter 1

Farrukh Azeem

10 Aug 2022

PONE-D-22-03287R1A genome-wide analysis of the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES (ASL/LBD) gene family in cassava (Manihot esculenta) and expression profile under biotic and abiotic stressPLOS ONE

Dear Dr. chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Sep 24 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Farrukh Azeem

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have corrected some of the things I pointed out. However, there are still many shortcomings. In addition, there are many inadequate understandings of important papers published in the past.

Although I suggested four papers to be concerned, the authors did not cite the three papers as follows. They should read precisely, cite them and add them in References.

Bowman et al., 2017. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell 171, 287-304 doi.org/10.1016/j.cell.2017.09.030

Machida Y., Suzuki T., Sasabe M., Iwakawa H., Kojima S., Machida C. Arabidopsis ASYMMETRIC LEAVES2 (AS2): roles in plant morphogenesis, cell division, and pathogenesis. J. Plant Research 2022 135:3–14 (doi.org/10.1007/s10265-021-01349-6)

Although I suggested that all AtLBD should be written AtASL/LBD in Figure 1, the authors wrote only 15 genes out of 43 genes as AtASL/LBD. They should change all AtLBD to AtASL/LBD.

Although I pointed, the authors did not change them in Figure 3. They should replace. C Block should be written ZF-motif, GAS block should be ICG/GAS-region, L-rich Block should be LZL-region.

Although the authors described that they could not achieve this analysis due to the timing given, they should understand precisely and cite the following two papers at least.

Chen WF, Wei XB, Rety S, Huang LY, Liu NN, Dou SX, Xi XG (2019) Structural analysis reveals a "molecular calipers" mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat. J Biol Chem 294:142–156

They did not complete to correct yet. They should correct all.</comment></comment></comment></comment></comment>

Reviewer #2: I have gone through the revised version of manuscript entitled "A genome-wide analysis of the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES (ASL/LBD) gene family in cassava (Manihot esculenta) and expression profile under biotic and abiotic stress" (PONE-D-22-03287R1). The authors have made satisfactory changes in the revised version of the manuscript. The manuscript in its current form can be ACCEPTED for publication in PlosONE.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr. Hamid Manzoor

**********

PLoS One. 2023 Apr 20;18(4):e0282100. doi: 10.1371/journal.pone.0282100.r004

Author response to Decision Letter 1

19 Sep 2022

Although I suggested four papers to be concerned, the authors did not cite the three papers as follows. They should read precisely, cite them and add them in References.

Bowman et al., 2017. Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome. Cell 171, 287-304 doi.org/10.1016/j.cell.2017.09.030

Response: Thanks for pointing out these omissions. Indeed, these references outstandingly studied the origin and evolution of the LOB domain. Therefore, these references were cited throughout the revised manuscript.

Although I suggested that all AtLBD should be written AtASL/LBD in Figure 1, the authors wrote only 15 genes out of 43 genes as AtASL/LBD. They should change all AtLBD to AtASL/LBD.

Response: Thanks for pointing it out. All AtLBD were changed to AtASL/LBD. Please see figure 1

Although I pointed, the authors did not change them in Figure 3. They should replace. C Block should be written ZF-motif, GAS block should be ICG/GAS-region, L-rich Block should be LZL-region.

Response: We apologize for this omission. C Block, GAS block, and L-rich Block were changed into ZF-motif, ICG/GAS-region, and LZL-region, respectively, throughout the manuscript and in figure 3. Please see figure 3.

Although the authors described that they could not achieve this analysis due to the timing given, they should understand precisely and cite the following two papers at least.

Response: Thanks for these suggestions. These references were cited throughout the revised manuscript.

They did not complete to correct yet. They should correct all.

Response: We apologize for omitting these important points. Efforts have been made to correct the misinformation. We hope our current revised manuscript meets the publication criteria in PlosOne.

Attachment

Submitted filename: Responses+To+Reviewers+11092022.docx

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PLoS One. doi: 10.1371/journal.pone.0282100.r005

Decision Letter 2

Vibhav Gautam

28 Nov 2022

PONE-D-22-03287R2A genome-wide analysis of the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES (ASL/LBD) transcription factors (TFs) in cassava ( Manihot esculenta ) and expression profile under biotic and abiotic stressPLOS ONE

Dear Dr. chen,

Please submit your revised manuscript by Jan 12 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Vibhav Gautam

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have corrected almost all. However, there are still ìnsuffícient part,<comment>

All AtLBD should be written AtASL/LBD, for example,

AtAS2/LBD6, AtASL1/LBD36, AtASL2/LBD10, AtASL3/LBD25, AtASL4/LOB, AtASL5/LBD12, AtASL6/LBD4, AtASL7/LBD11, AtASL8/LBD1, AtASL9/LBD3, AtASL10/LBD13, AtASL11/LBD15, AtASL12/LBD21, AtASL13/LBD24, AtASL14/LBD23, AtASL15/LBD17, AtASL16/LBD29, and so on.

See figure 4 and 5 in Machida et al., 2022 (https://link.springer.com/article/10.1007/s10265-021-01349-6).

Nomenclature with ASLs is important. This is because nomenclature with ASLs is based on the amino acid sequence homology of the AS2/LOB domain. Similar amino acid sequences have been shown to be functionally similar. Also in cassava, it will be possible to infer their functions by comparing with ASLs of Arabidopsis thaliana.

Two types of nomenclature (ASLs and LBDs in Arabidopsis thaliana) should be listed side by side and such nomenclature with ASLs should be advantageous for discussing relationships between the phylogeny and developmental functions of members in the AS2/LOB protein family. For example, at least four known proteins involved in auxin-induced lateral root formation are named ASL18/LBD16, ASL16/LBD29, ASL20/LBD18, and ASL24/LBD33; ASL15/LBD17 is also proposed to play a similar role. All of them are located in the close clades of sub-class Class Ia of A. thaliana in the phylogenetic tree. During pollen development, ASL1/LBD36, ASL2/LBD10, and ASL3/LBD25, all of which belong to the close narrow clades of the tree, control asymmetric cell division during pollen development. Interestingly, another set of ASL members (SCP/ASL29/LBD27 and ASL30/LBD22 in a small clade) are also involved in the same single process, but at different steps during the progression of pollen development. Note that ASL1/LBD36, ASL2/LBD10, and ASL3/LBD25 belong to Class Ia of A. thaliana, and that SCP/ASL29/LBD27 and ASL30/LBD22 belong to Class Ib of A. thaliana. Use of the ASL nomenclature might provide an edge to discussion of evolutionary developmental biology (evo-devo) of the AS2/LOB family.</comment>

Reviewer #3: The manuscript (PONE-D-22-03287) is well-prepared and quite conclusive. The language used in the manuscript is appropriate. The observations have been presented in a proper scientific manner and have been discussed well. Still, there is a scope to uplift the MS, I have a couple of suggestive comments which should further improve the manuscript.

Major comments

[1] The leaf lesion experiment in the VIGS lines is an important experiment, and currently, the images of the leaves provided are not very convincing. I encourage the authors to provide an image captured under similar light conditions (currently, the brightness and contrast of all the leaf areas are different). Also, Mark the inoculation point with a marker so that the inoculation site and lesion will be visible. Provide a picture of the entire leaf rather than a small section, then zoom in on the necrotic lesion area.

[2] Line#367 is there any information about these cultivators' abiotic and biotic stress sensitivity/tolerance? If yes, then provide that will strengthen this motive of the experiment.

Minor comments

[1] The discussion does not contain the figure references while mentioning the result, and please mention the figure number in the discussion too.

[2] Mention the statistical test performed while analyzing the data to test the significance in Figures 5 and 12

[3] Figure 6- Provide an explanation in the legend about the color code provided in the figure.

[4] Figure7- Specify tissue type and the variety of cassava used for transcription analysis as mentioned in the figure

[5] Figure 8- explain why the MeASLBD19 expression profile is missing (indicated by the gray color) in the disease group.

[6] Figure 9- The graph seems confusing; in MeASLBD12 and a few others, the relative expression value is significantly high at six h, but it still shows ns. This needs correction. Mention the statistical test performed.

[7] Fig 12b – provide scale bar.

Line#42 Gene expression is not a good keyword. Replace it.

Line#55, "development of fat symmetric," is this fat or flat?

Line#93 what is the basis of dividing into two subclasses? Class II is also divided into subclasses (a-g) which need to be mentioned.

Line#151. mentions other cassava cultivars, Arg7, W14, and KU50, that were used for gene analysis.

Line#155 Please specify how many leaves were inoculated per plant.

Line#156- Write bacterial dilution correctly using the superscript.

Line#156-Keep consistency in writing optical density here, it is OD600, while in line#194, it is written as (OD600).

Line#157- replace "hole" with "ring"

Line#162 - why only two similar replicates were considered?

Line#190-192 this explanation is not required, abridged.

Line#235 -Provide the hyperlink for the source code and web page for MCscanX.

Line#238 -Provide the hyperlink of the source for the Circos software

Line#256 provide the source website for the HISAT2 software.

Line#378 - RE-frame this sentence as "These results suggest the genotype-dependent tissue expression of these genes." Authors are using genotype and varieties as synonyms which is not the case. For example, in Line#382," cassava cultivars (Arg7 and W14)", these are referred to as Cultivars. However, KU50 and Arg7 are cultivated varieties and W14 wild ancestors. Correct this throughout the MS.

Line #382-Why the Variety KU50 not included in the expression analysis under drought treatment studies?

Line #385 "had significant differential expression under drought stress." Mention the fold change in expression compared to the control group.

Line# 427- "The 347 genes could be divided into four modules via WGCNA" explain the basis of this division into four modules indicated by different colors.

Reviewer #4: I found the paper to be well-prepared, with excellent and careful data selection, and it addressed all of the previous reviewers' comments with evidence. This MS could be useful for learning more about how ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES (ASL/LBD) transcription factors (TFs) in plants regulates biotic and abiotic stress responses. Therefore, this MS will timely provide important insight to understand ASL/LBD genes in plants under biotic and abiotic stress responses. However, main concerns associated with the MS are as follows:

(I)The authors have corrected most of the things suggested by previous reviewers: 1 and 2. However, I suggest to author please double check the AtLBD. It should be written AtASL/LBD in whole MS including figures and figure legends as also suggested by previous reviewers (twice).

(II)The figures and texts of Figure 1, Figure 2, Figure 3, Figure 10, and Figure S1 are unclear. I suggest authors should include high-resolution figures in their work to make it more understandable to a wider audience.

Reviewer #5: Mao and Abdoulaye et al - Genome-wide characterization of cassava LATERAL ORGAN BOUNDARIES domain genes revealed LBD47 involved in bacterial blight defense – PLOS ONE

The paper builds the study about genome-wide characterization of LBD genes using HMM profiles while establishing the role of MeLBD47 in plant phytohormone signalling in cassava. It showed significantly mitigated virulence of cassava bacterial blight (Xam CHN11) through Virus-induced gene silencing (VIGS).

General comment: The manuscript has the topic on the whole well covered and contains interesting set of experiments including VIGS and bioinformatic analyses that can be suitable for publication. Here are my few concerns:

Title: Please reframe the title of the paper. It does not sound very conclusive. One suggestion is “Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defense against bacterial blight”.

Line 23: ‘T’ small case in Arabidopsis thaliana

Line 32: ‘MeLBD47 was selected…’. How was it selected? Randomly? If yes, please mention that and if no, mention what was the basis of its selection for functional analyses?

Line 32 and 33: MeLBD47 is italicised somewhere and somewhere not. Please maintain uniformity.

Line 65: Add space between esculentaCrantz

Line 72: ‘extand’ misspelled

Line 99: 2-ΔCT or 2-ΔΔCT ?

Line 197-201: Information is repeated from Introduction.

Line 283: ‘leaf’, not ‘leave’

Line 343: ‘inoculated with’, not ‘Inoculated’

Discussion: Authors have mentioned in the introduction that this information will provide invaluable insights for further studies. I would suggest that the paper will be more informative if few lines are added in the discussion part about how this information can be put in use and what kinds of further studies can be done.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

PLoS One. 2023 Apr 20;18(4):e0282100. doi: 10.1371/journal.pone.0282100.r006

Author response to Decision Letter 2

1 Feb 2023

Dr. Yinhua Chen

Hainan Key Laboratory for Sustainable Utilisation of Tropical Bioresources,

College of Tropical Crops,

Hainan University,

Haikou 570228, Hainan, China

16.01.2023

Dear Editor-in-Chief,

With great pleasure, I re-submit the revised version of the manuscript ID PONE-D-22-03287 entitled "Genome-wide characterisation of cassava LATERAL ORGAN BOUNDARIES domain genes revealed LBD47 involved in bacterial blight defense" for consideration by PlosOne. Firstly, I would like to thank you for the opportunity to revise this manuscript. I highly appreciated the constructive comments and fruitful suggestions provided by each reviewer. Notably, some comments stemmed from the first round revised manuscript, probably because two reviewers were assigned to the first revision. Therefore, some comments were already addressed previously. The manuscript has certainly benefited from these insightful suggestions. Therefore, I look forward to working with you and the reviewers to move this manuscript for publication in the journal of PlosOne.

We have responded to all the reviewer's comments. Major improvements were made accordingly.

Reviewer #1: The authors have corrected almost all. However, there are still ìnsuffícient part,

All AtLBD should be written AtASL/LBD, for example,

See figure 4 and 5 in Machida et al., 2022 (https://link.springer.com/article/10.1007/s10265-021-01349-6).

Response: Thanks for this great point. The two terminology types were listed side by side (ASLs and LBDs). Please see figure 1

Response: Thanks for taking the time to read our manuscript and for all the constructive comments.

Major comments

Response: We apologize for the light conditions of the pictures. We have improved the picture with a scale bar, and hopefully, it is convincing enough. Please see picture 12

[2] Line#367 is there any information about these cultivators' abiotic and biotic stress sensitivity/tolerance? If yes, then provide that will strengthen this motive of the experiment.

Response: Thanks for these suggestions. Indeed, it could improve our manuscript. However, we haven’t conducted these analyses. And we apologize that we couldn’t conduct these analyses due to the time given.

Minor comments

[1] The discussion does not contain the figure references while mentioning the result, and please mention the figure number in the discussion too.

Response: Thanks for pointing these out. We have now referenced the figures in the revised manuscript. Please see the discussion section.

[2] Mention the statistical test performed while analyzing the data to test the significance in Figures 5 and 12

Response: We have now mentioned the statistical test performed. Please see figures 5 and 12

[3] Figure 6- Provide an explanation in the legend about the color code provided in the figure.

Response: We have now added an explanation about the color bar. Please see figure 6 legend

[4] Figure7- Specify tissue type and the variety of cassava used for transcription analysis as mentioned in the figure

Response: We Have now specified the tissue type and cassava varieties. Please see the figure 7 legend.

[5] Figure 8- explain why the MeASLBD19 expression profile is missing (indicated by the gray color) in the disease group.

Response: When we downloaded the expression profile data of cassava LBDs in diseases, we found that the expression abundance of MeASLBD19 was 0, therefore, it was displayed in gray.

Response: Thanks for pointing out these typos. It has now been revised. Please see figure 9

[7] Fig 12b – provide scale bar.

Response: Thanks; we have provided a scale bar. Please see figure 12b

Line#42 Gene expression is not a good keyword. Replace it.

Response: Thanks. Gene expression has been replaced with expression profiles. Please see line 41

Line#55, "development of fat symmetric," is this fat or flat?

Response: Thanks for pointing out this typo. It has been corrected. Please see line 54

Line#93 what is the basis of dividing into two subclasses? Class II is also divided into subclasses (a-g) which need to be mentioned.

Response: The tree was divided into two classes based on the clades form and previous studies that divided the arabidopsis LOD proteins into two classes: https://academic.oup.com/plphys/article/129/2/747/6110259#265176879

However, the subclasses (a-g) could not be mentioned because they resulted from the current studies.

Line#151. mentions other cassava cultivars, Arg7, W14, and KU50, that were used for gene analysis.

Response: Thanks for pointing it out. We have mentioned the missed cultivars. Please see 150

Line#155 Please specify how many leaves were inoculated per plant.

Response: It has been mentioned. Please see line 154

Line#156- Write bacterial dilution correctly using the superscript.

Response: Thanks for pointing it out. It has been corrected. Please see lines 155

Line#156-Keep consistency in writing optical density here, it is OD600, while in line#194, it is written as (OD600).

Response: We apologised for this inconsistency. We have corrected it with OD600 throughout the revised manuscript.

Line#157- replace "hole" with "ring"

Response: Thanks. We have replaced hole with ring. Please see line 156

Line#162 - why only two similar replicates were considered?

Response: We meant, “The experiments were conducted at least twice with identical results”.

Line#190-192 this explanation is not required, abridged.

Response: We have abridged the sentence. Please see line 138

Line#235 -Provide the hyperlink for the source code and web page for MCscanX.

Response: Thanks. The source code and web page have been provided. Please see line 235

Line#238 -Provide the hyperlink of the source for the Circos software

Response: Thanks. The hyperlink of the source for the circus software has been provided. Please see line 238

Line#256 provide the source website for the HISAT2 software.

Response: Thanks. The source website for the HISAT2 software has been provided. Please see line 257

Response: Thanks for pointing these out, and we apologise for the misleading. We have reframed the sentence. Please see lines 378-379

Besides, we have corrected these misleading sentences throughout the revised manuscript.

Line #382-Why the Variety KU50 not included in the expression analysis under drought treatment studies?

Response: Because the expression profiling data (downloaded from NCBI) did not contain the expression pattern of KU50 under drought.

Line #385 "had significant differential expression under drought stress." Mention the fold change in expression compared to the control group.

Response: Thanks for your great comment. Since the folds were not mentioned on the heat map, we think it might be better not to stay consistent with figure 8.

Line# 427- "The 347 genes could be divided into four modules via WGCNA" explain the basis of this division into four modules indicated by different colors.

Response: Highly interconnected gene sets were obtained by WGNCA analysis, and these gene sets were designated as "modules". A total of 4 modules were obtained. Among them, the gray module was considered a non-coexpressed gene set by default; therefore, it was not considered in the analysis. Please see lines 428-429

Response: Thanks for taking the time to read our manuscript and for all your suggestions. The two terminology types were listed side by side (ASLs and LBDs) throughout the revised manuscript.

Response: We apologise for the unclear pictures. We have replaced them with high-resolution throughout the revised manuscript.

Reviewer #5: Mao and Abdoulaye et al - Genome-wide characterization of cassava LATERAL ORGAN BOUNDARIES domain genes revealed LBD47 involved in bacterial blight defense – PLOS ONE

Response: Thanks for your comments and time to read our manuscript. The title has been reframed with the suggested title. Nevertheless, we would like to mention that most of the comments ref (Lines) were based on the first revised manuscript. Therefore we have already revised some parts previously.

Line 23: ‘T’ small case in Arabidopsis thaliana

Response: Thanks for pointing out this typo. It has been corrected. Please see line 21

Line 32: ‘MeLBD47 was selected…’. How was it selected? Randomly? If yes, please mention that and if no, mention what was the basis of its selection for functional analyses?

Response: MeLBD47 was selected based on the expression level under disease treatment and drought stress.

Line 32 and 33: MeLBD47 is italicised somewhere and somewhere not. Please maintain uniformity.

Response: We apologise for the inconsistency. We have revised it throughout the revised manuscript.

Line 65: Add space between esculentaCrantz

Response: It has been corrected

Line 72: ‘extand’ misspelled

Response: It has been corrected

Line 99: 2-ΔCT or 2-ΔΔCT ?

Response: It has been corrected

Line 197-201: Information is repeated from Introduction.

Response: It has been revised.

Line 283: ‘leaf’, not ‘leave’

Response: Thanks for pointing out these typos. It has been corrected throughout the revised manuscript.

Line 343: ‘inoculated with’, not ‘Inoculated’

Response: Thanks for pointing out these typos. It has been corrected throughout the revised manuscript.

Response: Thanks for these great suggestions. We have revised the discussion section accordingly, and hopefully, it meets the requirements.

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PLoS One. doi: 10.1371/journal.pone.0282100.r007

Decision Letter 3

Vibhav Gautam

8 Feb 2023

Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight

PONE-D-22-03287R3

Dear Dr. chen,

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PLoS One. doi: 10.1371/journal.pone.0282100.r008

Acceptance letter

Vibhav Gautam

10 Apr 2023

PONE-D-22-03287R3

Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight

Dear Dr. Chen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Data Availability Statement

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Genome-wide analyses of LATERAL ORGAN BOUNDARIES in cassava reveal the role of LBD47 in defence against bacterial blight

Yiming Mao

Assane Hamidou Abdoulaye

Jiming Song

Xiaowen Yao

Yijie Zhang

Yu Gao

Yiwei Ye

Kai Luo

Wei Xia

Yinhua Chen

Roles

Abstract

Introduction

Materials and methods

Plant material and growth conditions

RNA isolation, qRT-PCR analysis, and statistics analysis

Generation of pCsCMV VIGS construct and agroinfiltration

Identification and sequence analysis of MeASLBD genes

Phylogenetic tree construction, conserved motifs, and structure analysis

Chromosomal location, gene duplication event, gene collinearity, and synteny analysis

Cis-acting element analysis and identification of MeASLBDs target gene

Gene expression profiles of MeASLBDs in cassava

Co-expression analysis of MeASLBDs and identification of MeASLBD interacting genes by WGCNA

Results

Identification and chromosome localisation of cassava ASL/LBD genes

Evolutionary relationship and gene structure analysis of the MeASLBD genes

Fig 1. Phylogenetic analysis of AS2/LOB proteins in M. esculenta and A. thaliana.

Fig 2. Phylogenetic analysis of ASL/LBD genes and the structures and relative motif positions of the ASL/LBD gene family members in M. esculenta.

Conserved motif analysis of MeASLBD genes

Fig 3. Conserved domains of MeASLBD proteins.

Duplication, Collinearity, and synteny analysis

Fig 4. Chromosomal distribution and interchromosomal relationships of MeASLBD genes.

Fig 5. Colinearity analysis.

Analysis of putative cis-elements of the MeASLBD gene

Fig 6. The cis-elements of 2000 bp sequences upstream of MeASLBD gene analysed with the online tool PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html).

Expression analysis of MeASLBD genes in different tissues and organs

Fig 7. Expression pattern analysis of MeASLBD transcription factors in roots and leaves.

Expression profiles of the MeASLBD genes in response to drought and XamCHN11 infection

Fig 8. Expression profiles of MeASLBD genes under drought and XamCHN11 infection.

Validation of six MeASLBD genes in response to XamCHN11 via qRT-PCR

Fig 9. Expression patterns of 6 ASL/LBD genes in different samples of M. esculenta (1h, 3h, 6h, 9h, and CK).

Identification of candidate genes coexpressed with MeASLBD genes

Fig 10. GO enrichment analysis of MeASLBDs target genes.

Fig 11. The network of MeASLBDs and target genes in the turquoise module.

Silencing of MeASLBD47 reduced the lesion area and enabled differential expression of target genes

Fig 12. Silencing of MeASLBD47 in cassava plants using a cassava common mosaic virus (Cscmv)-based Virus-induced gene silencing (VIGS) system.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Farrukh Azeem

Roles

Author response to Decision Letter 0

Decision Letter 1

Farrukh Azeem

Roles

Author response to Decision Letter 1

Decision Letter 2

Vibhav Gautam

Roles

Author response to Decision Letter 2

Decision Letter 3

Vibhav Gautam

Roles

Acceptance letter

Vibhav Gautam

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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