Skip to main content
NCBI home page
Molecular Plant Pathology logoLink to Molecular Plant Pathology
. 2024 Jan 8;25(1):e13413. doi: 10.1111/mpp.13413

Infection‐specific transcriptional patterns of the maize pathogen Cochliobolus heterostrophus unravel genes involved in asexual development and virulence

Huilin Yu 1, Jiyue Zhang 1, Jinyu Fan 1, Wantong Jia 1, Yanan Lv 1, Hongyu Pan 1,✉, Xianghui Zhang 1,✉
PMCID: PMC10775821  PMID: 38279855

Abstract

Southern corn leaf blight (SCLB) caused by Cochliobolus heterostrophus is a destructive disease that threatens global maize (Zea mays) production. Despite many studies being conducted, very little is known about molecular processes employed by the pathogen during infection. There is a need to understand the fungal arms strategy and identify novel functional genes as targets for fungicide development. Transcriptome analysis based on RNA sequencing was carried out across conidia germination and host infection by C. heterostrophus. The present study revealed major changes in C. heterostrophus gene expression during host infection. Several differentially expressed genes (DEGs) induced during C. heterostrophus infection could be involved in the biosynthesis of secondary metabolites, peroxisome, energy metabolism, amino acid degradation and oxidative phosphorylation. In addition, histone acetyltransferase, secreted proteins, peroxisomal proteins, NADPH oxidase and transcription factors were selected for further functional validation. Here, we demonstrated that histone acetyltransferases (Hat2 and Rtt109), secreted proteins (Cel61A and Mep1), peroxisomal proteins (Pex11A and Pex14), NADPH oxidases (NoxA, NoxD and NoxR) and transcription factors (Crz1 and MtfA) play essential roles in C. heterostrophus conidiation, stress adaption and virulence. Taken together, our study revealed major changes in gene expression associated with C. heterostrophus infection and identified a diverse repertoire of genes critical for successful infection.

Keywords: asexual development, Cochliobolus heterostrophus, infection, transcriptome, virulence

Histone acetyltransferases, secreted proteins, peroxisomal proteins, NADPH oxidases and transcription factors play essential roles in Cochliobolus heterostrophus conidiation, stress adaption and host infection.

graphic file with name MPP-25-e13413-g002.jpg

1. INTRODUCTION

Southern corn leaf blight (SCLB) is one of the most significant factors constraining maize (Zea mays) production worldwide. Each year, despite the deployment of extensive use of fungicides and resistant varieties, about 10%–20% of maize harvest is lost due to SCLB (Balint‐Kurti et al., 2007; Wang et al., 2014). SCLB is caused by the necrotrophic fungus Cochliobolus heterostrophus, which infects leaves, sheaths and ear husks of maize (Chen et al., 2023). In 1970, a new virulent race (race T) appeared in Florida and swept up the east coast of the United States in a single growing season, destroying more than 15% of the maize crop (Condon et al., 2018). In the past decades, SCLB is still a major foliar disease of maize, and currently outbreaks in tropical and subtropical regions, such as the south‐eastern United States, Latin America and the Yellow Huai‐Hai River plain of China (Chen et al., 2023).

C. heterostrophus is a typical necrotrophic fungus, and mainly relies on conidia and mycelia to spread and infect in the field. Even though both conidia and mycelia of C. heterostrophus cause symptoms on maize leaves, conidia inoculation makes the penetration process occur much faster than if mycelia were inoculated. Upon conidial germination, the fungus does not grow far on the plant surface, while the germ tubes rapidly form appressoria, and penetration pegs breach the plant tissue. Faster penetration of hyphae emerging from germinating conidia suggests that the small appressoria of this fungus, although dispensable for infection, confer an advantage for rapid entry through the plant cuticle and epidermis (Lev & Horwitz, 2003). This means the conidial germination and appressoria formation are critical for infection in C. heterostrophus.

So far, many genes have been functionally identified by targeted gene deletion in C. heterostrophus, and most of them are related to T‐toxin production and virulence. The genomes of five C. heterostrophus strains were sequenced in 2013, namely, C5 (ATCC 48332, race O, MAT1‐1, Tox1 − ), C4 (ATCC 48331, race T, MAT1‐2, Tox1 +) and three field strains; several core secondary metabolism genes and small secreted protein candidate effector‐encoding genes have been identified (Condon et al., 2013). In Condon's research, the genes responsible for T‐toxin production were discovered, which included PKS1, PKS2, LAM1, OXI1, TOX9, TOX10, DEC1, RED1, RED2 and RED3 (Condon et al., 2018). After 5 years, through PacBio long‐read sequencing, Haridas et al. revealed the Tox1 gene arrangement and the breakpoints in C4 and corresponding positions in race T are large race T‐specific insertions (Haridas et al., 2023). A nonribosomal peptide synthetase‐encoding gene NPS6 is responsible for the biosynthesis of extracellular siderophores and a virulence determinant in C. heterostrophus (Oide et al., 2006). A Lae1‐like methyltransferase, Llm1, was identified in C. heterostrophus that acts as a negative regulator of T‐toxin production and impacts virulence on the host (Bi et al., 2013). NPS6 is regulated by a GATA transcription factor siderophore biosynthesis repressor Sre1 (Zhang et al., 2013). These studies have provided insights into the biology of C. heterostrophus, especially the ability of the fungus to produce T‐toxin. However, there are many aspects of the biology of C. heterostrophus that need investigation. For example, in addition to toxins, what other factors are involved in virulence in C. heterostrophus? How does C. heterostrophus overcome host defence? C. heterostrophus must acquire carbon and nitrogen sources to fuel its growth, but how this is achieved is not clear. In contrast to gene functional studies, one of the main reasons for the current lack of understanding is the lack of systematic investigation of plant infection.

In this study, we aimed to identify major changes in gene expression to define the transcriptional landscape during C. heterostrophus infection. We also aimed to identify the full repertoire of fungal virulence determinants deployed by C. heterostrophus.

2. RESULTS

2.1. C. heterostrophus exhibited great transcriptional variability during host infection

To examine the transcriptional landscape during infection by C. heterostrophus, we inoculated 3‐week‐old B73 maize detached leaves with a 20 μL drop of conidial suspension of 2 × 105 spores/mL. B73 is a maize inbred line with moderate susceptibility to C. heterostrophus that was used as a host in our previous study (Yu et al., 2022). The inoculated maize leaves were collected for sample extraction at 12 h and 24 h post‐inoculation, and 7‐day‐old C. heterostrophus CMX cultures were used as 0 h control. These two time points covered conidial germination, appressorium formation, appressorium‐mediated penetration, invasive hypha proliferation and cell‐to‐cell movement. Using Illumina sequencing of mRNA libraries extracted from maize tissues, 61.48 Gb clean data were generated from nine samples. Kraken 2 was used to separate C. heterostrophus reads from the mixed transcriptome, and here we only focus on reads that mapped to the annotated C. heterostrophus genome (Wood et al., 2019). Feature Counts v. 1.5.0‐p3 was used to count the reads numbers mapped to each gene. Then, the fragments per kilobase million (FPKM) of each gene were calculated based on the length of the gene and the number of reads mapped to the respective gene. Principal component analysis (PCA) was conducted with FPKM to test the reproducibility between biological replicates of each sample (Figure 1a). In addition, Pearson's correlation coefficient test showed that there was a good correlation among the biological replicates (Figure 1b).

FIGURE 1.

FIGURE 1

Open in a new tab

Overview of transcriptome data sets of Cochliobolus heterostrophus infection. (a) Principal component analysis of the transcriptome data sets. (b) Pearson correlation coefficient of the transcriptome data sets. (c) Venn diagram showing a comparison of differentially expressed genes (DEGs) from three comparisons. (d–f) Volcano plot of the distribution of DEGs in three comparison groups, 12 hours post‐inoculation (hpi) versus 0 hpi (d), 24 hpi versus 0 hpi (e) and 24 hpi versus 12 hpi (f). The blue colour and red colour represent the significantly down‐regulated genes and up‐regulated genes, respectively, while the grey colour indicates no significant expression. The x axis represents the change in gene expression in different samples, here represented by log2[fold change] value. The y axis indicates the statistical significance of changes in gene expression levels, here represented by −log10(p adj) value.

In total, 9386 differentially expressed genes (DEGs) were identified in three comparison groups. At 12 hours post‐inoculation (hpi) versus 0 hpi, 4868 DEGs were identified, including 1975 up‐regulated genes and 2893 down‐regulated genes (Figure 1c). In 24 hpi versus 0 hpi, 1413 genes were up‐regulated and 1105 genes were down‐regulated, respectively (Figure 1d). Compared with 12 hpi, 1246 up‐regulated genes and 754 down‐regulated genes were identified at 24 hpi (Figure 1e). A Venn diagram shows a comparison of DEGs from the three comparisons (Figure 1f). These prominent changes in gene expression inferred great transcriptional variability of C. heterostrophus during infection.

In order to analyse the dynamic changes of some specific genes during infection by C. heterostrophus, we identified genes that were co‐expressed during the infection process using a Venn diagram of co‐expression trend (Figure S1). According to the results of hierarchical clustering, there were four stage‐specific gene expression patterns identified. The heat map shows the hierarchical clustering of DEGs in these patterns, with patterns ranging in size from 331 to 843, and gene expression trends differing in each pattern (Figure 2a). For example, Pattern 1 (P1) contains 843 genes whose expression peaks appeared in conidia and that were down‐regulated at 12 and 24 hpi. These genes included genes involved in fungal sporulation and signal transduction, such as the gene VOS1 coding for the velvet family member vos1, and the catalase gene CAT1. Histidine kinase‐encoding gene NIK1 and MAP kinase kinase kinase (MAPKKK) gene SSK2 were also grouped in P1. This is consistent with previous studies on the regulation of conidial production by sensing external signals (Figure 2b). The genes related to early fungal infection were mainly assigned to pattern P2; their expression peaks appeared at 12 hpi. These genes included NPS6, RED1, RTT109, and PEX1. NPS6 encodes a nonribosomal peptide synthetase involved in siderophore‐mediated iron metabolism, RED1 is necessary for T‐toxin production, both genes are indispensable for virulence of C. heterostrophus. Histone acetyltransferase‐encoding gene RTT109 and peroxisome gene PEX1 have been reported to play an important role in the virulence of Magnaporthe oryzae and Fusarium graminearum (Deng et al., 2016; Kong et al., 2018; Kwon et al., 2018; Zhang et al., 2019) (Figure 2c). Pattern P3 contains 334 genes, which include exoglucanase‐encoding gene GUX1 and FAD‐linked oxidoreductase‐encoding gene ZEB1; both genes were significantly up‐regulated at 24 hpi (Figure 2d). Several genes were grouped in pattern P4, including CUT1 (cerase‐encoding gene), XYN11A (xylanase‐encoding gene), CHS1 (chitin synthetase‐encoding gene) and CYP51 (sterol synthesis‐related gene). All these genes were induced at 12 and 24 hpi (Figure 2e).

FIGURE 2.

FIGURE 2

Open in a new tab

Stage‐specific temporal expression of the Cochliobolus heterostrophus transcriptome during infection. (a) Heat map showing the hierarchical clustering of pathogen genes from each co‐expression pattern. (b) Relative expression for representative genes in pattern 1 (P1). (c) Relative expression for representative genes in pattern 2 (P2). (d) Relative expression for representative genes in pattern 3 (P3). (e) Relative expression for representative genes in pattern 4 (P4). hpi, hours post‐inoculation.

To determine the physiological processes controlled by co‐expressed genes in each pattern, we performed a KEGG enrichment analysis. As shown in Figure 3a, during the early stage of infection, the pathogen needs to proliferate quickly and pathways including ribosome biogenesis and RNA polymerase are significantly enriched in P2. The high expression of genes related to fatty acid and other glycan degradation (P3) may be necessary for fungi to consume fatty acids and glycogen to provide energy for mycelial expansion in the cell during the late stage of infection. During invasive growth, starch and sucrose metabolism and pentose interconversions are significantly enriched in P4. In addition, compared with the conidial stage (P1), the expression levels of peroxisome pathway‐related genes were increased in P2–P4.

FIGURE 3.

FIGURE 3

Open in a new tab

Special pathways involved in infection by Cochliobolus heterostrophus. (a) KEGG enrichment analysis of genes in each pattern using clusterProfiler reveals over‐represented physiological functions during C. heterostrophus infection. (b) The peroxisome pathway was significantly up‐regulated at 12 and 24 hours post‐inoculation (hpi). (c) Hierarchical clustering of transcriptional levels of the PEX genes. (d) PPI network prediction of PEX genes during C. heterostrophus infection.

As a supplement to KEGG enrichment analysis, we determined the expression levels of all peroxisome pathway genes at each time point. The z‐score was used for normalization, and the expression levels at 12 and 24 hpi were compared with the conidial stage. We found the peroxisome pathway was significantly up‐regulated at the infection stage (Figure 3b). A heat map showed the hierarchical clustering of fungal genes encoding proteins associated with presumed peroxisome biosynthesis and transport (Figure 3c). Through PPI network prediction of DEGs in the above pathways, we found that the protein Pex14 may play a key regulatory role (Figure 3d).

GO term enrichment analysis for P1 to P4 revealed the biological processes and molecular functions involved in the asexual reproduction and infection by C. heterostrophus. The integral component of membrane, channel activity and protein histidine kinase activity were enriched in P1 (Figure 4a). This suggested that virulence factors such as transcription factors or cell wall‐degrading enzymes play a major role in fungal infection. There were also considerable enrichments of other GO terms associated with the intracellular expansion of fungal invasive mycelia, such as histone modification (P2), cofactor binding, glutathione metabolic process (P3), peptidase activity and hydrolase activity (P4) (Figure 4a). In the conidial stage (P1), there were some transmembrane proteins with relatively low expression during infection, which function as membrane components or ion channels, including probable sulphate permease (COCC4DRAFT_77247), sugar transporter Stl1 (COCC4DRAFT_77587) and malic acid transport protein (COCC4DRAFT_128747) (Figure 4b). During the early fungal infection stage (P2), the expression levels of several transcription factors were up‐regulated, among which forkhead family transcript factor Fkh2 (COCC4DRAFT_183321) has been reported to be essential for virulence in a variety of plant‐pathogenic fungi (Figure 4c). Some membrane proteins, such as polyol transporter (COCC4DRAFT_44727) and lactose permease (COCC4DRAFT_183287), were also enriched in P3 (Figure 4c). The enrichment pathway of peptidase activity and hydrolase activity in P4 included many genes encoding putative secreted proteins, such as polysaccharide monooxygenase Cel61A (COCC4DRAFT_53511), endo‐1,4‐β‐xylanase Xyn2 (COCC4DRAFT_59433) and extracellular metalloproteinase Mep1 (COCC4DRAFT_67491) (Figure 4e). These genes may play a key role in fungal infection. In conclusion, during the process of asexual reproduction and infection of C. heterostrophus, the overall transcription level undergoes significant changes. These results encouraged us to investigate the roles of DEGs in C. heterostrophus infection.

FIGURE 4.

FIGURE 4

Open in a new tab

GO term enrichment analysis for four patterns (P1 to P4) revealed the biological processes and molecular functions involved in the asexual reproduction and infection of Cochliobolus heterostrophus. hpi, hours post‐inoculation.

2.2. Identification of up‐regulated genes in C. heterostrophus during infection

2.2.1. Peroxins encoding genes

Peroxins, encoded by PEX genes, are essential factors for peroxisome biogenesis in various species from yeast to humans (Okumoto et al., 2020). In our transcriptomic analysis, the expression levels of 15 PEX genes were changed in C. heterostrophus during infection, and 12 of them were up‐regulated: PEX1 (COCC4DRAFT_39736), PEX2 (COCC4DRAFT_122608), PEX3 (COCC4DRAFT_79387), PEX4 (COCC4DRAFT_200011), PEX6 (COCC4DRAFT_131845), PEX7 (COCC4DRAFT_206750), PEX10 (COCC4DRAFT_22752), PEX11A (COCC4DRAFT_1681806), PEX11B (COCC4DRAFT_44584), PEX12 (COCC4DRAFT_166382), PEX14 (COCC4DRAFT_168796) and PEX19 (COCC4DRAFT_150311) (Figure 3c). In addition, the expression levels of PEX3, PEX6, PEX11A, PEX12 and PEX14 were determined using reverse transcription‐quantitative PCR (RT‐qPCR), and we found that they were significantly induced at 12 hpi (Figure S2). These results suggested that PEX genes may play an important role in virulence in C. heterostrophus.

2.2.2. Genes encoding histone acetyltransferase

Three genes encoding histone acetyltransferases, HAT1 (COCC4DRAFT_196767), HAT2 (COCC4DRAFT_58008) and RTT109 (COCC4DRAFT_189576), were up‐regulated in C. heterostrophus during infection (Figure S3a). These results encouraged us to investigate the roles of histone acetyltransferases in C. heterostrophus infection.

2.2.3. Secreted proteins

Plant pathogens, especially biotrophic and hemibiotrophic pathogens, secrete proteins during colonization to establish a successful pathogen–host interaction. C. heterostrophus is a typical necrotrophic fungus, and many secreted protein‐encoding genes were up‐regulated during infection, which included CEL61A (COCC4DRAFT_53511), MEP1 (COCC4DRAFT_67491) and some CFEM domain‐containing protein‐encoding genes (COCC4DRAFT_89751, COCC4DRAFT_65556, COCC4DRAFT_48195). This made us very interested in the functions of secreted proteins in necrotrophic fungi.

2.2.4. NADPH oxidase

NADPH oxidases (Noxs) are flavoenzymes that function by transferring electrons across biological membranes to catalyse the reduction of molecular oxygen to superoxide (Sumimoto, 2008). In filamentous fungi, Noxs are necessary for cellular differentiation and virulence (Heller & Tudzynski, 2011; Takemoto et al., 2007, 2011). Noxs are necessary for septin‐mediated reorientation of the F‐actin cytoskeleton to facilitate cuticle rupture and plant cell invasion in rice blast fungus (Ryder et al., 2013). In C. heterostrophus, four NOX genes were identified: ChNOXA (COCC4DRAFT_179971), ChNOXB (COCC4DRAFT_83933), ChNOXD (COCC4DRAFT_151652) and ChNOXR (COCC4DRAFT_81448). All four genes were induced during infection; ChNOXA, ChNOXD and ChNOXR showed continuous up‐regulation at 12 and 24 hpi, whereas ChNOXB was only up‐regulated at 12 hpi (Figure S3b). These results indicate that Noxs may be necessary for early and late infection stages.

2.2.5. Transcription factors

Transcription factors (TFs), such as C2H2‐type zinc‐finger TF, forkhead box TF, bZIP TF, and bHLH TF, play key regulatory roles in phytopathogenic fungi. In this study, two C2H2‐type zinc‐finger TFs, MtfA (COCC4DRAFT_123864) and Crz1 (COCC4DRAFT_144568), were induced at 12 and 24 dpi. In addition, a fungal‐specific TF (COCC4DRAFT_205122), which contains a fungal TF regulatory middle homology region domain, was also up‐regulated during infection by C. heterostrophus (Figure S3c). These findings suggest that TFs may be critical for fungal infection and colonization in C. heterostrophus.

2.3. Histone acetyltransferase‐encoding genes ChRTT109 and ChHAT2 are involved in asexual development and virulence in C. heterostrophus

To investigate the functions of histone acetyltransferase‐encoding genes in transcriptomic analysis, ChRTT109, ChHAT1 and ChHAT2 were deleted. To our surprise, in contrast to the wild type (WT), no significant development or virulence reduction was detected for Δhat1 mutant strains, suggesting that ChHAT1 is dispensable for development and virulence in C. heterostrophus (data not shown). Knockout of ChRTT109 resulted in a significant reduction in vegetative growth (Figure 5a,b). The number of conidia produced by Δhat2 mutant strains was reduced significantly compared to the numbers produced by the WT strain (Figure 3c). In addition, compared with the WT, Δrtt109 and Δhat2 mutant strains showed increased sensitivity to H2O2 (Figure 5a,d). Additionally, compared with the WT, both Δrtt109 and Δhat2 mutant strains caused smaller lesions on maize (Figure 5e,f). These results indicated that the reduced virulence of Δrtt109 and Δhat2 mutant strains may be related to increased sensitivity to H2O2, and ChHAT2 not ChHAT1 played important roles in development and virulence in C. heterostrophus. However, ChRTT109, ChHAT1 and ChHAT2 were not involved in the response to nitrosative stress (Figure 5d).

FIGURE 5.

FIGURE 5

Open in a new tab

Histone acetyltransferase is required for mycelial growth, conidiation and virulence in Cochliobolus heterostrophus. (a) Growth characteristics of wild type (WT) and mutant strains on complete medium with xylose (CMX) without or with different concentrations of sodium nitroprusside (SNP) and H2O2. (b) Statistical analysis of colony diameter of indicated strains on CMX. (c) Statistical analysis of conidiation of indicated strains. (d) Statistical analysis of mycelial growth of indicated strains on CMX with different concentrations of SNP and H2O2. (e) Δrtt109 and Δhat2 mutants exhibited decreased virulence on detached maize leaves. (f) Statistical analysis of lesion size caused by indicated strains where the centre line indicates the median percentiles. GraphPad Prism program's t tests and multiple t test were used for the analysis of significant differences. The bars indicate standard error of the mean, and asterisks represent significant difference (*p < 0.05, **p < 0.01).

2.4. ChCEL61A and ChMEP1 are required for conidiation and virulence in C. heterostrophus

In this study, several secreted protein‐encoding genes were up‐regulated during infection by C. heterostrophus, including CEL61A and MEP1. To further explore the roles of CEL61A and MEP1 in C. heterostrophus, the mutants were constructed. As shown in Figure 6a, only Δmep1 mutant strains exhibited a significantly slower mycelial growth rate on a complete medium with xylose (CMX) (Figure 6a,b). In addition, Δmep1 mutant strains produced more green/black aerial hyphae than the WT (Figure 6a). Generally, as conidia are the source of dark green/black pigmentation of the WT, the darker colouration of mutant hyphae reflects more conidiation. Consistent with the hyphal colour, the numbers of conidia produced by Δmep1 mutant strains were at least 7 times more than the WT (Figure 6c). In contrast to Δmep1 mutant strains, conidial numbers of Δcel61A mutant strains were reduced drastically compared with the WT (Figure 6c). However, neither ChMEP1 nor ChCEL1 was necessary for nitrosative and oxidative stress adaption in C. heterostrophus (Figure 6a,d). In addition, inoculation of Δmep1 or Δcel61A mutant strains caused smaller lesions on detached maize leaves (Figure 6e,f).

FIGURE 6.

FIGURE 6

Open in a new tab

ChCEL61A and ChMEP1 are required for conidiation and virulence in Cochliobolus heterostrophus. (a) Growth characteristics of wild type (WT) and indicated mutant strains on complete medium with xylose (CMX) without or with different concentrations of sodium nitroprusside (SNP) and H2O2. (b) Statistical analysis of colony diameter of indicated strains on CMX. (c) Statistical analysis of conidiation of indicated strains. (d) Statistical analysis of mycelial growth of indicated strains on CMX with different concentrations of SNP and H2O2. (e) Δcel61A and Δmep1 mutants exhibited decreased virulence on detached maize leaves. (f) Statistical analysis of lesion size caused by indicated strains where the centre line indicates the median percentiles. GraphPad Prism program's t tests and multiple t test were used for the analysis of significant differences. The bars indicate standard error of the mean, and asterisks represent significant difference (*p < 0.05, **p < 0.01).

2.5. ChPEX11 and ChPEX14 are necessary for development, stress adaptation and virulence in C. heterostrophus

Previous reports revealed that nitric oxide (NO) plays a critical role in the host immunity of plants (Jian et al., 2021; Yun et al., 2016). Hosts perceiving pathogens can provoke reactive nitrogen species (RNS) derived from NO and superoxide, leading to nitrosative stress (Arasimowicz‐Jelonek & Floryszak‐Wieczorek, 2016; Di Pietro & Talbot, 2017). As mentioned in 2.2.1, several PEX genes were up‐regulated during infection in C. heterostrophus, deletion of ChPEX11A and ChPEX14 caused increased sensitivity to sodium nitroprusside (SNP) (Figure 7a,d). In addition, Δpex14 mutant strains exhibited slower mycelial growth, more conidiation and increased sensitivity to H2O2 (Figure 7a–d). Moreover, detached maize leaves inoculated with Δpex11A and Δpex14 mutants showed significantly smaller lesions (Figure 7e,f). These observations suggest that deletion of ChPEX11A and ChPEX14 leads to increased sensitivity of mutants to oxidative and nitrosative stress, which resulted in decreased virulence in C. heterostrophus.

FIGURE 7.

FIGURE 7

Open in a new tab

ChPEX11 and ChPEX14 are necessary for development, stress adaptation and virulence in Cochliobolus heterostrophus. (a) Growth characteristics of wild type (WT) and indicated mutant strains on complete medium with xylose (CMX) without or with different concentrations of sodium nitroprusside (SNP), H2O2 and calcofluor white (CFW). (b) Statistical analysis of colony diameter of indicated strains on CMX. (c) Statistical analysis of conidiation of indicated strains. (d) Statistical analysis of mycelial growth of indicated strains on CMX medium with different concentrations of SNP, H2O2 and CFW. (e) Δpex11A and Δpex14 mutants exhibited decreased virulence on detached maize leaves. (f) Statistical analysis of lesion size caused by indicated strains where the centre line indicates the median percentiles. GraphPad Prism program's t tests and multiple t test were used for the analysis of significant differences. The bars indicate standard error of the mean, and asterisks represent significant difference (*p < 0.05, **p < 0.01).

2.6. NADPH oxidases are involved in cell wall biosynthesis, asexual and sexual development, stress adaption and virulence in C. heterostrophus

NADPH oxidases (Noxs) are the most common enzymes that are responsible for the production of reactive oxygen species (ROS). Noxs have been extensively studied in fungi in the recent years. We deleted the C. heterostrophus NADPH oxidase‐encoding genes in the WT C4 using homologous gene recombination. The normal hyphal growth rate was observed in ΔnoxA, ΔnoxD and ΔnoxR mutants, and colonies displayed lighter pigmentation on CMX. However, ΔnoxA, ΔnoxD and ΔnoxR mutants exhibited a slower growth rate on minimal medium (MM) (Figure 8a,b). In addition, ΔnoxA, ΔnoxD and ΔnoxR mutants showed a dramatic reduction in conidiation (Figure 8c). We questioned whether Noxs also function in stress adaption in C. heterostrophus. Mycelial plugs from WT, ΔnoxA, ΔnoxD and ΔnoxR mutants were inoculated onto CMX containing 10 mM H2O2, 30 mM menadione, 50 μg/mL calcofluor white (CFW), 300 μg/mL Congo red (CR) or 0.25 μg/mL tebuconazole and their sensitivity was evaluated after 7 days. Compared to the WT, the ΔnoxA, ΔnoxD and ΔnoxR mutants showed increased sensitivity to 10 mM H2O2, 30 mM menadione and 0.25 μg/mL tebuconazole, but ΔnoxA and ΔnoxD mutants were more resistant to 50 μg/mL CFW and 300 μg/mL CR (Figure 8a,d). CFW preferentially binds to polysaccharides containing 1,4‐linked d‐glucopyranosyl units and therefore alters the assembly of chitin microfibrils in fungi (Elorza et al., 1983), so sensitivity to CFW is related to the chitin content of cell walls, and NoxA and NoxD are necessary for correct cell wall biosynthesis in C. heterostrophus. Moreover, the pigmented apothecia formation was significantly affected in ΔnoxA, ΔnoxD and ΔnoxR mutants (Figure 8e,f). These results indicated that Noxs are not only involved in asexual development but also sexual development in C. heterostrophus. Importantly, ΔnoxA, ΔnoxD and ΔnoxR mutants exhibited significantly reduced virulence in maize (Figure 8g,h).

FIGURE 8.

FIGURE 8

Open in a new tab

NADPH oxidases are involved in cell wall biosynthesis, asexual and sexual development, stress adaption and virulence in Cochliobolus heterostrophus. (a) Growth characteristics of wild type (WT) and indicated mutant strains on complete medium with xylose (CMX), minimal medium (MM) and CMX amended with H2O2, menadione, calcofluor white (CFW), Congo red (CR) and tebuconazole. (b) Statistical analysis of colony diameter of indicated strains on CMX and MM. (c) Statistical analysis of conidiation of indicated strains. (d) Statistical analysis of mycelial growth of indicated strains on CMX amended with H2O2, menadione, CFW, CR and tebuconazole. (e) Deletion of ChNoxA, ChNoxD and ChNoxR affected the sexual development of C. heterostrophus. (f) Statistical analysis of pseudothecia formation of indicated strains. (g) ΔnoxA, ΔnoxD and ΔnoxR mutants exhibited decreased virulence on detached maize leaves. (h) Statistical analysis of lesion size caused by indicated strains where the centre line indicates the median percentiles. GraphPad Prism program's t tests and multiple t test were used for the analysis of significant differences. The bars indicate standard error of the mean, and asterisks represent significant difference (*p < 0.05, **p < 0.01).

2.7. A C2H2‐type transcription factor Crz1 is indispensable for stress adaption and virulence in C. heterostrophus

To further investigate the role of TFs in the development and virulence of C. heterostrophus, two up‐regulated TFs Crz1 and MtfA were deleted. After 7 days' growth on CMX, the colonies of the Crz1 and MtfA knockout mutants showed similar growth rates with the WT (Figure 9a,b). However, SNP at different concentrations significantly reduced the growth of the Crz1 knockout mutants compared to the WT (Figure 9a,d). In contrast, Crz1 knockout mutants produced at least three times the number of conidia than the WT strain on CMX (Figure 9c). Further observation showed that the Crz1 knockout mutants caused a much smaller lesion size on detached leaves (Figure 9e,f). The decreased virulence of Crz1 knockout mutants may be related to the increased sensitivity to SNP. However, deletion of MtfA only caused reduced conidiation in C. heterostrophus (Figure 9c).

FIGURE 9.

FIGURE 9

Open in a new tab

Transcription factors are indispensable for conidiation, stress adaption and virulence in Cochliobolus heterostrophus. (a) Growth characteristics of wild type (WT) and indicated mutant strains on complete medium with xylose (CMX) without or with different concentrations of sodium nitroprusside (SNP) and H2O2. (b) Statistical analysis of colony diameter of indicated strains on CMX. (c) Statistical analysis of conidiation of indicated strains. (d) Statistical analysis of inhibitory rate of indicated strains on CMX amended with different concentrations of SNP and H2O2. (e) ΔCrz1 mutants exhibited decreased virulence on detached maize leaves. (f) Statistical analysis of lesion size caused by indicated strains where the centre line indicates the median percentiles. GraphPad Prism program's t tests and multiple t test were used for the analysis of significant differences. The bars indicate the standard error of the mean, and asterisks represent the significant difference (*p < 0.05, **p < 0.01).

3. DISCUSSION

In this study, we investigated the infection‐specific transcriptional patterns of C. heterostrophus, a destructive pathogen of maize and a representative of the largest taxonomic group of fungal plant pathogens, the Dothideomycetes. The ability of the C. heterostrophus to colonize plant tissue and cause disease is still relatively poorly understood, and many molecular mechanisms underlying their infection cycles remain unknown. Many studies have provided insight into the roles of individual genes in virulence, but systematic analysis of transcriptional profile during infection in C. heterostrophus has been seldom uncovered (Condon et al., 2018; Yu et al., 2022; Zhang et al., 2020). The aim of this study was to reveal fundamental changes in gene expression during infection through transcriptional profiling to identify the virulence‐related genes deployed by C. heterostrophus.

Several transcriptomic analyses have been conducted previously to investigate plant–microbe interactions to explore the underlying mechanism of fungal development in host plants (Dobon et al., 2016; Kawahara et al., 2012; Kleemann et al., 2012; Lanver et al., 2018; Rudd et al., 2015; Wang et al., 2017; Yan et al., 2023). Most of these studies focused on hemibiotrophic or biotrophic fungi and provided significant insight into the biology of biotrophic development. In a recent study on C. heterostrophus using RNA‐seq analysis, the majority of DEGs were identified as being associated with mitochondrial, cell wall and chitin synthesis, sugar metabolism, antibiotics and carbon metabolism (Meshram et al., 2022). However, the samples were collected at 48 h post‐inoculation, at which time symptoms have already started appearing on the susceptible maize line. In addition, the roles of up‐regulated genes during infection were not investigated (Meshram et al., 2022). As the first 24 h are critical for C. heterostrophus infection, we decided to perform a comprehensive transcriptional analysis at this early stage of infection, to identify the maximum number of fungal transcripts. Based on the expression data of C. heterostrophus genes during conidial germination and host infection, a total of 9386 DEGs in three comparison groups were identified. This reflected great diversity and specificity in mRNA levels during the process of infection.

During the infection, 3388 genes were specifically up‐regulated, which included glycoside hydrolase‐encoding genes, histone acetyltransferase‐encoding genes, NADPH oxidase‐encoding genes, TFs and peroxisomal protein‐encoding genes. Among them, the most induced were glycoside hydrolase‐encoding genes. Several genes encoding glycoside hydrolase were up‐regulated more than 100 times during C. heterostrophus infection. C. heterostrophus is a typical necrotrophic fungus, and glycoside hydrolases are critical for degrading host cell walls and disturbing plant immunity. In the maize smut fungus Ustilago maydis, Erc1 binds to host cell wall components and displays 1,3‐β‐glucanase activity, which is required to attenuate β‐glucan‐induced defence responses. In addition, Erc1 has a cell type‐specific virulence function, which is necessary for fungal cell‐to‐cell extension in the plant bundle sheath; this function is fully conserved in the Erc1 orthologue of the barley pathogen Ustilago hordei (Ökmen et al., 2022). Through proteomic analysis, a glycoside hydrolase 12 family protein Fg05851 was identified in F. graminearum, and two paralogous genes were highly induced during infection and had important roles in F. graminearum virulence during infection (Wang et al., 2023). Ma et al. identified the GH12 protein XEG1 from Phytophthora sojae, which exhibits xyloglucanase activity and promotes infection by degrading plant cell walls (Ma et al., 2015). In addition, a COPII cargo receptor MoErv29 is required for the delivery of secreted protein MoSlp1, and both MoErv29 and MoSlp1 are indispensable for infection (Qian et al., 2022). Sta1 is a secreted effector identified in the biotrophic fungus U. maydis, and sta1 mutants exhibit a dramatic reduction of virulence (Tanaka et al., 2020). All these results revealed that secreted proteins are conserved and necessary for virulence in fungi and oomycetes. In future, we will investigate the pathogenesis of glycoside hydrolase in C. heterostrophus and explore the immune receptors responsible for the innate immune perception of microbial pathogens in maize.

Chromatin composed of nucleosomes condenses to form chromosomes during cell division, and the core of the nucleosome is four histone proteins H2A, H2B, H3 and H4. The histones are involved in many cellular processes through histone N‐terminal modification, such as acetylation, phosphorylation, methylation and ubiquitination (Yin et al., 2019). Histone acetyltransferases (HATs) are responsible for the acetylation of histones on specific lysine residues and are critical for DNA replication, transcriptional activation, cell cycle regulation and gene silencing (Grienenberger et al., 2002; Reifsnyder et al., 1996). Recent studies have shown that HATs participate in virulence in fungi. Two B‐type HATs (FgHat1 and FgHat2) have been identified in F. graminearum, deletion FgHAT2 not FgHAT1 resulted in severely defective vegetative growth, conidia production, deoxynivalenol (DON) biosynthesis and virulence (Luv et al., 2020). In M. oryzae, MoHat1 is important for appressorial penetration and pathogenicity. However, the other B‐type gene MoHAT2 has not been functionally characterized in M. oryzae. Here, Hat1 and Hat2 were also identified in C. heterostrophus; only ChHAT2 was induced during infection. ChHAT2 was found to be necessary for conidiation and virulence, whereas targeted deletion of ChHAT1 did not result in any detectable phenotypes. Therefore, we speculate that only one Hat is required for virulence in phytopathogenic fungi. In our study, another acetyltransferase Rtt109 was characterized, and Δrtt109 mutant strains exhibited defects in conidiation, stress adaption and virulence. This was consistent with previous studies on M. oryzae and Beauveria bassiana (Cai et al., 2018; Kwon et al., 2018).

One of the first responses of plants to microbial attack is the production of extracellular superoxide surrounding infection sites. Some phytopathogenic fungi undergo an oxidative burst of their own during plant infection, which is associated with the development of specialized infection structures (Egan et al., 2007). In M. oryzae, scavenging of oxygen radicals significantly delays the development of appressoria and alters their morphology (Egan et al., 2007). NADPH oxidases are responsible for producing ROS, Δnox1 and Δnox2 mutants are incapable of causing plant disease in M. oryzae (Egan et al., 2007). In addition, Noxs are necessary for septin‐mediated reorientation of the F‐actin cytoskeleton to facilitate cuticle rupture and plant cell invasion in M. oryzae (Ryder et al., 2013). The catalytic subunits NoxA, B and D have been shown to be involved in fruiting body formation, sclerotia formation, appressoria‐mediated penetration and virulence (Lara‐Ortiz et al., 2003; Ryder et al., 2013; Schürmann et al., 2013; Siegmund et al., 2015). The regulatory subunit NoxR did not influence the expression of NoxB, but negatively regulated NoxA, and ΔnoxR displayed an increased sensitivity to H₂O₂ and many ROS‐generating oxidants in Alternaria alternata (Yang & Chung, 2013). In the current study, ChNoxA, ChNoxD and ChNoxR were also found to be involved in virulence in C. heterostrophus. C. heterostrophus differs from M. oryzae as appressoria are not necessary for infection by C. heterostrophus. We do not know if the Noxs are associated with the formation of appressoria in C. heterostrophus. Further work is needed to unravel the detailed mechanism of Noxs in C. heterostrophus virulence.

Besides the secreted proteins, histone acetyltransferases and NADPH oxidase, many other kinds of factors, such as peroxins and TFs, are critical for infection in C. heterostrophus. All these results reflect great transcriptional variability during C. heterostrophus host infection and our study provides a foundation for further gene functional annotation in C. heterostrophus and SCLB management.

4. EXPERIMENTAL PROCEDURES

4.1. Fungal strains, plant materials and growth conditions

Zea mays inbred line B73 (susceptible to C. heterostrophus) was cultured in a growth chamber at 25°C with 16 h light. C. heterostrophus WT strain C4 (Tox1 +, MAT1‐2, ATCC 48331) grown on a complete medium with xylose (CMX) was used for inoculation.

A conidial suspension of C. heterostrophus was collected from 7‐day‐old CMX plates and adjusted to 200 conidia/μL. A 20 μL of conidial suspension was inoculated on 3‐week‐old B73 maize detached leaves, and inoculated samples were collected after 12 and 24 h.

4.2. RNA extraction and sequencing

Total RNA was extracted using TRIzol reagent (Invitrogen), and RNA integrity was evaluated using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). Only samples with an RNA Integrity Number (RIN) >7 were used to construct the sequencing libraries. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly‐T oligo‐attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in First‐Strand Synthesis Reaction Buffer (5×). First‐strand cDNA was synthesized using a random hexamer primer and M‐MuLV reverse transcriptase. Second‐strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, adaptors with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter), and library quality was assessed on the Agilent Bioanalyzer 2100 system.

The clustering of the index‐coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v. 3‐cBot‐HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired‐end reads were generated. Clean reads were obtained by removing reads containing adapter, reads containing poly‐N and low‐quality reads from raw data. All the downstream analyses were based on the clean reads with high quality. The mapped reads of each sample were assembled by StringTie (v. 1.3.3b) in a reference‐based approach. The FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene, and differential expression analysis was performed using the DESeq2 R package. Corrected p‐value of 0.05 and absolute fold change of 2 were set as the threshold for significantly differential expression. Gene Ontology (GO) enrichment analysis of DEGs was performed using the R package clusterProfiler, and p < 0.05 was considered significantly enriched with regard to DEGs. We used the R package clusterProfiler to test the statistical enrichment of DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

4.3. Mutant construction and PCR verification

The target gene deletion was conducted using the split‐marker method as described previously (Zhang et al., 2020). In brief, the 5′ and 3′ flanking regions of each gene were amplified with primer pair FP1/RP1 and FP2/RP2, and fused with hygromycin resistance cassette (HPH) amplified from plasmid pUCATPH (Lu et al., 1994) with primer pair M13F/M13R. The resulting fusion fragments were transformed into protoplasts of WT strain C4 to replace the target gene by homologous recombination, and transformants were screened on CMX without salts supplemented with hygromycin B. All the deletion mutants were verified by diagnostic PCR with primers listed in Table S1. For each gene, at least two independent deletion mutants were chosen for further analysis. The previously published method was used for mutant complementation (Zhang et al., 2020).

4.4. Phenotypic analysis

To determine the mycelial growth rate of WT and mutant strains, 5‐mm‐diameter mycelial plugs were inoculated on CMX and incubated in the dark at 25°C. After 7 days, the colony diameters of WT and mutant strains were measured and recorded. To evaluate the sensitivity of the WT and mutant strains to stress, mycelial plugs of each strain were placed on CMX supplemented with H2O2 (5 and 10 mM), sodium nitroprusside (5 and 10 mM SNP) and calcofluor white (CFW, 50 μg/mL), Congo red (300 μg/mL) and tebuconazole (0.25 μg/mL). All assays were repeated three times.

To quantify conidial production, 7‐day‐old cultures of each strain on CMX were dislodged with sterile water, then filtered through four layers of cheesecloth to remove mycelial debris. The number of conidia was counted using a haemocytometer under microscopic examination. For sexual reproduction, WT strain C4 (MAT1‐2) or mutants and the albino WT strain CB7 (MAT1‐1, alb1) were used for crosses following standard crossing protocols (Zhang et al., 2020). After 21 days, the average number of albino and pigmented pseudothecia per square centimetre on maize leaf was counted to evaluate the ability of pseudothecia to form. Four replicates were evaluated for each strain.

4.5. Virulence assays

The maize inbred line B73, which is susceptible to C. heterostrophus C4, was used for the virulence test. A conidial suspension was obtained as described above and diluted to 100 conidia/μL. Detached maize leaves were inoculated with 20 μL conidial suspension and were transferred to a Petri dish and kept for 24 h, then were moved out and kept at 25°C under 16 h of light/8 h of dark. Lesion size was determined at 72 hpi, and the virulence test was repeated three times.

4.6. Statistical analysis

For statistical analysis, GraphPad Prism program's t tests and multiple t tests were used for the analysis of significant differences. All data shown are the mean ± standard error of the median (SEM).

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

Supporting information

FIGURE S1. Venn diagram showing co‐expressed genes in the infection process of Cochliobolus heterostrophus. To define the overall dynamic changes in transcript levels, we analysed gene expression using hierarchical clustering to identify expression patterns. For this Venn diagram, each circle represents the number of genes having expression levels (FPKM > 1) in a group. The overlapping region of the circles indicates the number of co‐expressed genes between the corresponding multiple groups, also non‐overlapping regions represent specifically expressed genes.

Click here for additional data file. (8MB, docx)

FIGURE S2. Relative expression levels of PEX genes during Cochliobolus heterostrophus infection. Reverse transcription‐quantitative PCR was used to detect the relative expression levels of five PEX genes during different developmental stages, and ACTIN was used as an endogenous control. GraphPad Prism program’s t tests and multiple t test were used for the analysis of significant differences. The bars indicate the standard error of the mean, and asterisks represent the significant difference (*p < 0.05, **p < 0.01).

Click here for additional data file. (1.1MB, docx)

FIGURE S3. Hierarchical clustering of the expression of genes predicted to encode histone acetyltransferases (a), NADPH oxidases (b) and transcription factors (c) during Cochliobolus heterostrophus infection.

Click here for additional data file. (2.8MB, docx)

TABLE S1. Primers used in this study.

Click here for additional data file. (34.7KB, docx)

ACKNOWLEDGEMENTS

This research was funded by the Project of International Collaboration Plan in Jilin Province (20210402035GH).

Yu, H. , Zhang, J. , Fan, J. , Jia, W. , Lv, Y. , Pan, H. et al. (2024) Infection‐specific transcriptional patterns of the maize pathogen Cochliobolus heterostrophus unravel genes involved in asexual development and virulence. Molecular Plant Pathology, 25, e13413. Available from: 10.1111/mpp.13413

Contributor Information

Hongyu Pan, Email: panhongyu@jlu.edu.cn.

Xianghui Zhang, Email: zhangxianghui1982@126.com.

DATA AVAILABILITY STATEMENT

Transcriptome data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1043471 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1043471).

REFERENCES

  1. Arasimowicz‐Jelonek, M. & Floryszak‐Wieczorek, J. (2016) Nitric oxide in the offensive strategy of fungal and oomycete plant pathogens. Frontiers in Plant Science, 7, 252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balint‐Kurti, P.J. , Zwonitzer, J.C. , Wisser, R.J. , Carson, M.L. , Oropeza‐Rosas, M.A. , Holland, J.B. et al. (2007) Precise mapping of quantitative trait loci for resistance to southern leaf blight, caused by Cochliobolus heterostrophus race O, and flowering time using advanced intercross maize lines. Genetics, 176, 645–657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bi, Q. , Wu, D.L. , Zhu, X.D. & Turgeon, B.G. (2013) Cochliobolus heterostrophus Llm1 – a Lae1‐like methyltransferase regulates T‐toxin production, virulence, and development. Fungal Genetics and Biology, 51, 21–33. [DOI] [PubMed] [Google Scholar]
  4. Cai, Q. , Wang, J.J. , Shao, B. , Ying, S.H. & Feng, M.G. (2018) Rtt109‐dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana . Pest Management Science, 74, 2626–2635. [DOI] [PubMed] [Google Scholar]
  5. Chen, G.S. , Xiao, Y.J. , Dai, S. , Dai, Z.K. , Wang, X.M. , Li, B.L. et al. (2023) Genetic basis of resistance to southern corn leaf blight in the maize multi‐parent population and diversity panel. Plant Biotechnology Journal, 21, 506–520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Condon, B.J. , Elliott, C. , González, J.B. , Yun, S.H. , Akagi, Y. , Wiesner‐Hanks, T. et al. (2018) Clues to an evolutionary mystery: the genes for T‐toxin, enabler of the devastating 1970 southern corn leaf blight epidemic, are present in ancestral species, suggesting an ancient origin. Molecular Plant‐Microbe Interactions, 31, 1154–1165. [DOI] [PubMed] [Google Scholar]
  7. Condon, B.J. , Leng, Y.Q. , Wu, D.L. , Bushley, K.E. , Ohm, R.A. , Otillar, R. et al. (2013) Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens. PLoS Genetics, 9, e1003233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deng, S. , Gu, Z. , Yang, N. , Li, L. , Yue, X. , Que, Y. et al. (2016) Identification and characterization of the peroxin 1 gene MoPEX1 required for infection‐related morphogenesis and pathogenicity in Magnaporthe oryzae . Scientific Reports, 6, 36292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Di Pietro, A. & Talbot, N.J. (2017) Fungal pathogenesis: combatting the oxidative burst. Nature Microbiology, 2, 17095. [DOI] [PubMed] [Google Scholar]
  10. Dobon, A. , Bunting, D.C. , Cabrera‐Quio, L.E. , Uauy, C. & Saunders, D.G. (2016) The host pathogen interaction between wheat and yellow rust induces temporally coordinated waves of gene expression. BMC Genomics, 17, 380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Egan, M.J. , Wang, Z.Y. , Jones, M.A. , Smirnoff, N. & Talbot, N.J. (2007) Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. Proceedings of the National Academy of Sciences of the United States of America, 104, 11772–11777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elorza, M.V. , Rico, H. & Sentandreu, R. (1983) Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. Journal of General Microbiology, 129, 1577–1582. [DOI] [PubMed] [Google Scholar]
  13. Grienenberger, A. , Miotto, B. , Sagnier, T. , Cavalli, G. , Schramke, V. , Geli, V. et al. (2002) The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Current Biology, 12, 762–766. [DOI] [PubMed] [Google Scholar]
  14. Haridas, S. , González, J.B. , Riley, R. , Koriabine, M. , Yan, M. , Ng, V. et al. (2023) T‐toxin virulence genes: unconnected dots in a sea of repeats. mBio, 25, e0026123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heller, J. & Tudzynski, P. (2011) Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annual Review of Phytopathology, 49, 369–390. [DOI] [PubMed] [Google Scholar]
  16. Jian, Y.Q. , Liu, Z.Y. , Wang, H.X. , Chen, Y. , Yin, Y.N. , Zhao, Y.F. et al. (2021) Interplay of two transcription factors for recruitment of the chromatin remodeling complex modulates fungal nitrosative stress response. Nature Communications, 12, 2576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kawahara, Y. , Oono, Y. , Kanamori, H. , Matsumoto, T. , Itoh, T. & Minami, E. (2012) Imultaneous RNA‐seq analysis of a mixed transcriptome of rice and blast fungus interaction. PLoS One, 7, 49423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kleemann, J. , Rincon‐Rivera, L.J. , Takahara, H. , Neumann, U. , Loren, V. , van Themaat, E. et al. (2012) Sequential delivery of host‐induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum . PLoS Pathogens, 8, e1002643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kong, X.J. , Diepeningen, A.D.V. , Lee, T.A.J.V.D. , Waalwijk, C. , Xu, J.S. , Xu, J. et al. (2018) The Fusarium graminearum histone acetyltransferases are important for morphogenesis, DON biosynthesis, and pathogenicity. Frontiers in Microbiology, 9, 654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kwon, S. , Lee, J. , Jeon, J. , Kim, S. , Park, S.Y. , Jeon, J. et al. (2018) Role of the histone acetyltransferase Rtt109 in development and pathogenicity of the rice blast fungus. Molecular Plant‐Microbe Interactions, 31, 1200–1210. [DOI] [PubMed] [Google Scholar]
  21. Lanver, D. , Muller, A.N. , Happel, P. , Schweizer, G. , Haas, F.B. & Franitza, M. (2018) The biotrophic development of Ustilago maydis studied by RNA‐Seq analysis. The Plant Cell, 30, 300–323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lara‐Ortiz, T. , Riveros‐Rosas, H. & Aguirre, J. (2003) Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans . Molecular Microbiology, 50, 1241–1255. [DOI] [PubMed] [Google Scholar]
  23. Lev, S. & Horwitz, B.A. (2003) A mitogen‐activated protein kinase pathway modulates the expression of two cellulase genes in Cochliobolus heterostrophus during plant infection. The Plant Cell, 15, 835–844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lu, S. , Lyngholm, L. , Yang, G. , Bronson, C. , Yoder, O.C. & Turgeon, B.G. (1994) Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme‐mediated integration. Proceedings of the National Academy of Sciences of the United States of America, 91, 12649–12653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Luv, W.Y. , Yang, N. , Xu, Z. , Dai, H. , Tang, S. & Wang, Z.Y. (2020) FgHAT2 is involved in regulating vegetative growth, conidiation, DNA damage repair, DON production and virulence in Fusarium graminearum . Journal of Integrative Agriculture, 19, 1813–1824. [Google Scholar]
  26. Ma, Z.C. , Song, T.Q. , Zhu, L. , Ye, W.W. , Wang, Y. , Shao, Y.Y. et al. (2015) A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP. The Plant Cell, 27, 2057–2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meshram, S. , Gogoi, R. , Bashyal, B.M. , Kumar, A. , Mandal, P.K. & Hossain, F. (2022) Comparative transcriptome analysis of fungal pathogen Bipolaris maydis to understand pathogenicity behavior on resistant and susceptible non‐CMS maize genotypes. Frontiers in Microbiology, 13, 837056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oide, S. , Moeder, W. , Krasnoff, S. , Gibson, D. , Haas, H. , Yoshioka, K. et al. (2006) NPS6, encoding a nonribosomal peptide synthetase involved in siderophore‐mediated iron metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. The Plant Cell, 18, 2836–2853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ökmen, B. , Jaeger, E. , Schilling, L. , Finke, N. , Klemd, A. , Lee, Y.J. et al. (2022) A conserved enzyme of smut fungi facilitates cell‐to‐cell extension in the plant bundle sheath. Nature Communications, 13, 6003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Okumoto, K. , Tamura, S. , Honsho, M. & Fujiki, Y. (2020) Peroxisome: metabolic functions and biogenesis. Advances in Experimental Medicine and Biology, 1299, 3–17. [DOI] [PubMed] [Google Scholar]
  31. Qian, B. , Su, X.T. , Ye, Z.Y. , Liu, X.Y. , Liu, M.X. , Shen, D.Y. et al. (2022) MoErv29 promotes apoplastic effector secretion contributing to virulence of the rice blast fungus Magnaporthe oryzae . New Phytologist, 233, 1289–1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reifsnyder, C. , Lowell, J. , Clarke, A. & Pillus, L. (1996) Yeast SAS silencing genes and human genes associated with AML and HIV‐1 tat interactions are homologous with acetyltransferases. Nature Genetics, 14, 42–49. [DOI] [PubMed] [Google Scholar]
  33. Rudd, J.J. , Kanyuka, K. , Hassani‐Pak, K. , Derbyshire, M. , Andongabo, A. & Devonshire, J. (2015) Transcriptome and metabolite profiling of the infection cycle of Zymoseptoria tritici on wheat reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions and a variation on the hemibiotrophic lifestyle definition. Plant Physiology, 167, 1158–1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ryder, L.S. , Dagdas, Y.F. , Mentlak, T.A. , Kershaw, M.J. , Thornton, C.R. , Schuster, M. et al. (2013) NADPH oxidases regulate septin‐mediated cytoskeletal remodeling during plant infection by the rice blast fungus. Proceedings of the National Academy of Sciences of the United States of America, 110, 3179–3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schürmann, J. , Buttermann, D. , Herrmann, A. , Giesbert, S. & Tudzynski, P. (2013) Molecular characterization of the NADPH oxidase complex in the ergot fungus Claviceps purpurea: CpNox2 and CpPls1 are important for a balanced host–pathogen interaction. Molecular Plant‐Microbe Interactions, 26, 1151–1164. [DOI] [PubMed] [Google Scholar]
  36. Siegmund, U. , Marschall, R. & Tudzynski, P. (2015) BcNoxD, a putative ER protein, is a new component of the NADPH oxidase complex in Botrytis cinerea . Molecular Microbiology, 95, 988–1005. [DOI] [PubMed] [Google Scholar]
  37. Sumimoto, H. (2008) Structure, regulation and evolution of Nox‐family NADPH oxidases that produce reactive oxygen species. FEBS Journal, 275, 3249–3277. [DOI] [PubMed] [Google Scholar]
  38. Tanaka, S. , Gollin, I. , Rössel, N. & Kahmann, R. (2020) The functionally conserved effector Sta1 is a fungal cell wall protein required for virulence in Ustilago maydis . New Phytologist, 227, 185–199. [DOI] [PubMed] [Google Scholar]
  39. Takemoto, D. , Kamakura, S. , Saika, S. , Becker, Y. , Wrenn, R. , Tanaka, A. et al. (2011) Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex. Proceedings of the National Academy of Sciences of the United States of America, 108, 2861–2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takemoto, D. , Tanaka, A. & Scott, B. (2007) NADPH oxidases in fungi: diverse roles of reactive oxygen species in fungal cellular differentiation. Fungal Genetics and Biology, 44, 1065–1076. [DOI] [PubMed] [Google Scholar]
  41. Wang, X. , Zhang, Y. , Xu, X. , Li, H. , Wu, X. , Zhang, S. et al. (2014) Evaluation of maize inbred lines currently used in Chinese breeding programs for resistance to six foliar diseases. Crop Journal, 2, 213–222. [Google Scholar]
  42. Wang, Z.D. , Yan, N. , Wang, Z.H. , Zhang, X.H. , Zhang, J.Z. & Xue, H.M. (2017) RNA‐seq analysis provides insight into reprogramming of culm development in Zizania latifolia induced by Ustilago esculenta . Plant Molecular Biology, 95, 533–547. [DOI] [PubMed] [Google Scholar]
  43. Wang, Z.K. , Yang, B. , Zheng, W.Y. , Wang, L. , Cai, X.X. , Yang, J. et al. (2023) Recognition of glycoside hydrolase 12 proteins by the immune receptor RXEG1 confers Fusarium head blight resistance in wheat. Plant Biotechnology Journal, 21, 769–781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wood, D.E. , Lu, J. & Langmead, B. (2019) Improved metagenomic analysis with Kraken2. Genome Biology, 20, 257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yan, X. , Tang, B.Z. , Ryder, L.S. , MacLean, D. , Were, V.M. , Eseola, A.B. et al. (2023) The transcriptional landscape of plant infection by the rice blast fungus Magnaporthe oryzae reveals distinct families of temporally co‐regulated and structurally conserved effectors. The Plant Cell, 35, 1360–1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yang, S.L. & Chung, K.R. (2013) Similar and distinct roles of NADPH oxidase components in the tangerine pathotype of Alternaria alternate . Molecular Plant Pathology, 14, 543–556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yin, Z.Y. , Chen, C. , Yang, J. , Feng, W.Z. , Liu, X.Y. , Zuo, R.F. et al. (2019) Histone acetyltransferase MoHat1 acetylates autophagy‐related proteins MoAtg3 and MoAtg9 to orchestrate functional appressorium formation and pathogenicity in Magnaporthe oryzae . Autophagy, 15, 1234–1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yu, H. , Jia, W. , Xiao, K. , Jiao, W. , Zhang, X. & Pan, H. (2022) The autophagy genes ChATG4 and ChATG8 are required for reproductive development, virulence, and septin assembly in Cochliobolus heterostrophus . Phytopathology, 112, 830–841. [DOI] [PubMed] [Google Scholar]
  49. Yun, B.W. , Skelly, M. , Yin, M.H. , Yu, M.D. , Mun, B.G. , Lee, S.U. et al. (2016) Nitric oxide and S‐nitrosoglutathione function additively during plant immunity. New Phytologist, 211, 516–526. [DOI] [PubMed] [Google Scholar]
  50. Zhang, L. , Liu, C. , Wang, L. , Sun, S. , Liu, A. , Liang, Y. et al. (2019) FgPEX1 and FgPEX10 are required for the maintenance of Woronin bodies and full virulence of Fusarium graminearum . Current Genetics, 65, 1383–1396. [DOI] [PubMed] [Google Scholar]
  51. Zhang, N. , NurAinIzzati, M.Z. , Scher, K. , Condon, B.J. , Horwitz, B.A. & Turgeon, B.G. (2013) Iron, oxidative stress, and virulence: roles of iron‐sensitive transcription factor Sre1 and the redox sensor ChAp1 in the maize pathogen Cochliobolus heterostrophus . Molecular Plant‐Microbe Interactions, 26, 1473–1485. [DOI] [PubMed] [Google Scholar]
  52. Zhang, X.H. , González, J.B. & Turgeon, B.G. (2020) Septins are required for reproductive propagule development and virulence of the maize pathogen Cochliobolus heterostrophus . Fungal Genetics and Biology, 135, 103291. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

FIGURE S1. Venn diagram showing co‐expressed genes in the infection process of Cochliobolus heterostrophus. To define the overall dynamic changes in transcript levels, we analysed gene expression using hierarchical clustering to identify expression patterns. For this Venn diagram, each circle represents the number of genes having expression levels (FPKM > 1) in a group. The overlapping region of the circles indicates the number of co‐expressed genes between the corresponding multiple groups, also non‐overlapping regions represent specifically expressed genes.

Click here for additional data file. (8MB, docx)

FIGURE S2. Relative expression levels of PEX genes during Cochliobolus heterostrophus infection. Reverse transcription‐quantitative PCR was used to detect the relative expression levels of five PEX genes during different developmental stages, and ACTIN was used as an endogenous control. GraphPad Prism program’s t tests and multiple t test were used for the analysis of significant differences. The bars indicate the standard error of the mean, and asterisks represent the significant difference (*p < 0.05, **p < 0.01).

Click here for additional data file. (1.1MB, docx)

FIGURE S3. Hierarchical clustering of the expression of genes predicted to encode histone acetyltransferases (a), NADPH oxidases (b) and transcription factors (c) during Cochliobolus heterostrophus infection.

Click here for additional data file. (2.8MB, docx)

TABLE S1. Primers used in this study.

Click here for additional data file. (34.7KB, docx)

Data Availability Statement

Transcriptome data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1043471 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1043471).

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

RESOURCES