Identification and characterization of maize ACD6-like gene reveal ZmACD6 as the maize orthologue conferring resistance to Ustilago maydis

ABSTRACT

Enhancing broad-spectrum resistance is a major goal of crop breeding. However, broad-spectrum resistance has not been thoroughly investigated, and its underlying molecular mechanisms remain elusive. In the model plant Arabidopsis (Arabidopsis thaliana), ACCELERATED CELL DEATH6 (ACD6) is a key component of broad-spectrum resistance that acts in a positive feedback loop with salicylic acid (SA) to regulate multiple pattern recognition receptors. However, the role of ACD6 in disease resistance in crop plants is unclear. Here, we show that the transcript of ANK23, one of the 15 ACD6-like genes in maize (Zea mays), is induced by SA and by infection with the pathogenic fungus Ustilago maydis. Heterologous expression of ANK23 restored disease resistance in the Arabidopsis mutant acd6-2. We show that ANK23 is a maize ortholog of ACD6 and therefore rename ANK23 as ZmACD6. Furthermore, using CRISPR/Cas9, we generated ZmACD6 knockout maize plants, which are more susceptible to U. maydis than wild-type plants. We also identified a maize line (SC-9) with relatively high ZmACD6 expression levels from a diverse natural maize population. SC-9 has increased disease resistance to U. maydis and defense activation, suggesting a practical approach to cultivate elite varieties with enhanced disease resistance.

Introduction

Crop diseases reduce crop yield and quality and threaten global food security. Therefore, there is an urgent need to develop crop varieties with broad-spectrum resistance.¹ To achieve this, it is necessary to understand the underlying molecular mechanisms of broad-spectrum resistance in crops. Two layers of defense mechanisms against pathogens have evolved in plants, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI).² PTI can be triggered through the interactions between pattern recognition receptors (PRRs) and PAMPs.³ PRRs are receptor-like kinases found on the cell surface. In Arabidopsis (Arabidopsis thaliana), three PRRs have been characterized: FLAGELLIN SENSING2 (FLS2), EF-Tu receptor (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1).^4–6 Upon activation, these pathways share certain common signaling events, including accumulation of reactive oxygen species (ROS), mitogen-activated protein kinase activation, and callose deposition.⁷

PTI is relatively mild and is the main component of plant basal resistance, contributing to broad-spectrum resistance. By contrast, ETI is resistance (R) gene specific, stronger, and often triggers the hypersensitivity response (HR) at the infection site. In turn, the HR induces the development of broad-spectrum resistance in uninfected distant tissues, in a process termed systemic acquired resistance (SAR).⁸

The small phenolic compound salicylic acid (SA) is an essential signal molecule for plant defense against a broad spectrum of pathogens. Extensive studies in Arabidopsis have shown that SA plays multiple roles in the regulation of plant defense, including basal and R-mediated defense as well as SAR.⁹ As a regulator of SA-mediated pathways, the Arabidopsis ACCELERATED CELL DEATH6 (ACD6) acts in a positive feedback loop with SA to confer enhanced resistance.^10,11 The dominant gain-of-function mutant acd6-1 shows a constitutive autoimmunity phenotype, including high levels of SA, small stature, spontaneous cell death, mitogen-activated protein kinase activation, increased callose accumulation, and enhanced resistance to pathogens.^10–14

Moreover, natural alleles of ACD6 from different Arabidopsis accessions partially phenocopy acd6-1 and confer enhanced resistance to a broad spectrum of pathogens.¹⁵ By contrast, the T-DNA mutant of ACD6 is more susceptible to Pseudomonas syringae and shows delayed SA accumulation during pathogen infection.¹⁰ ACD6 is a multipass transmembrane protein with an N-terminal cytosolic ankyrin repeat domain.^11,14 Ankyrin-containing proteins act as a scaffold in protein–protein interactions.¹⁶ Indeed, an analysis of ACD6 complexes revealed that ACD6 interacts with multiple PRRs and are part of the ACD6/SA feedback loop.^14,17 Thus, ACD6 is a critical component in the defense response against a broad spectrum of pathogens.

Maize (Zea mays) is an example of a monocotyledonous plant with a fully sequenced genome that has been adopted as a model to study the molecular mechanism of the defense response in cereal crops. However, in contrast to the extensive work conducted on disease resistance in Arabidopsis, little is known about SA-mediated defense in maize. The maize genome contains 15 ACD6-like genes.¹⁸ However, whether there is a maize ortholog of ACD6 is unknown.

In this study, we identified and characterized ANK23 as the maize ortholog of AtACD6. ZmACD6 knockout plants generated using the CRISPR/Cas9 system exhibited decreased disease resistance to Ustilago maydis, whereas a maize line (SC-9) expressing relatively high ZmACD6 levels identified from a natural population showed increased disease resistance to U. maydis and enhanced activation of downstream SA signaling. These results suggest that ZmACD6 is a positive modulator of disease resistance that functions by regulating the SA pathway.

Results

ZmANK23 is induced upon SA treatment and U. maydis infection

The maize genome has 15 ACD6-like homologs that encode transmembrane proteins containing ankyrin repeats.¹⁸ A phylogenetic analysis indicated that ZmANK37 is the closest member of the maize family to AtACD6 (Figure 1). ACD6 functions as a regulator and effector in a positive feedback loop with SA;¹⁰ therefore, we further examined whether the expression of all 15 ACD6-like genes is induced by SA. Three established SA-responsive genes, ZmPR4, ZmPR5, and ZmPR10, were used as positive controls.¹⁹ After treating the plants with SA, we observed that ZmANK23 was the only member that was strongly induced by SA, as determined by quantitative real-time PCR (qPCR) (Figure 2, Supplementary Figure S1). Therefore, we hypothesized that ZmANK23 functions as an ortholog of AtACD6 in maize.

Figure 1.

Phylogenetic analysis of Arabidopsis ACD6 and ankyrin transmembrane proteins in maize. The phylogenetic tree was constructed using the neighbor-joining method, and bootstrap values from 1000 replicates for each branch are listed. The scale bar indicates 0.1.

Figure 2.

Time-course induction of ZmANK23 by SA. qPCR analysis of maize leaves treated with 200 μΜ SA revealed induction of SA-associated PRs (a–c) and ZmANK23 expression (d). All expression levels were normalized to ACTIN. The bars indicate the standard error of three replicates for each sample analyzed together. This experiment was repeated three times with similar results. P-values were calculated by Student’s t-test (*P < .05; ***P < .001).

As AtACD6 expression was previously shown to be systemically induced during pathogen infection,¹⁰ we predicted that ZmANK23 would also be induced by pathogens. U. maydis is a biotrophic fungal plant pathogen that induces prominent disease symptoms on all aerial parts of maize and the U. maydis–maize pathosystem is often used as a model system.²⁰ We infected maize B73 plants with U. maydis strain SG200. SG200 is an engineered solopathogenic haploid strain that can infect maize without a mating partner.²¹ As expected, the ZmPR10 transcript levels were increased upon infection (Figure 3). Furthermore, ZmANK23 transcript levels were strongly increased at 4 days after SG200 infection (Figure 3). Thus, ZmANK23 is a SA- and pathogen-induced gene.

Figure 3.

ZmANK23 expression during pathogen infection. Maize seedlings were infected with biotrophic U. maydis wild-type strain SG200. Infected leaves were analyzed by qPCR at the indicated time points. This experiment was repeated three times with similar results. P-values were calculated by Student’s t-test (***P < .001).

ZmANK23 is a functional ACD6 gene

The acd6-2 loss-of-function plants displayed modestly increased susceptibility to the Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000).¹⁵ To determine whether ZmANK23 could complement the Arabidopsis acd6-2 mutant, we introduced ZmANK23 cDNA clones into the acd6-2 background. Five independent transformants carrying at least one copy of ZmANK23 had restored resistance to Pst DC3000 (Figure 4, Supplementary Figure S2A). This restored resistance in plants heterologously expressing ZmACD6 was accompanied by a decrease in the severity of disease symptoms induced by Pst DC3000 (Supplementary Figure S2B). Therefore, ZmANK23 can be considered as the maize ACD6 ortholog and is hereafter referred to as ZmACD6.

Figure 4.

Complementation of the Arabidopsis loss-of-function mutant acd6-2 by ZmANK23. P. syringae growth curve. Col, acd6-2, and acd6-2 plants transformed with ZmANK23 (acd6-2/ZmANK23) were infected with Pst DC3000 at an OD₆₀₀ = 0.0001. Bars indicate standard errors (n = 6). This experiment was repeated three times with similar results. cfu, colony-forming units.

ZmACD6 knockout maize plants have reduced resistance to U. maydis

To analyze and confirm the contribution of ZmACD6 to disease resistance in maize, we used the CRISPR/Cas9-mediated genome editing technique to knock out ZmACD6. Two knockout alleles were generated. The first allele, Zmacd6-1, carries a 5-nucleotide deletion, while Zmacd6-2 has a 1-nucleotide insertion (Figure 5(a)). Both result in a frameshift near the single-guide RNA target site (Figure 5(a)). These lines were used to test disease resistance to U. maydis SG200. Symptoms were more severe in the ZmACD6 CRISPR lines than in the wild-type KN5585 line (Figure 5(b,c)). Therefore, ZmACD6 is important for maize resistance to U. maydis.

Figure 5.

CRISPR/Cas9-mediated knockout of ZmACD6 confers increased susceptibility to U. maydis. (a) The genomic structures of ZmACD6 (GRMZM2G123977) carrying the 5-bp deletion or 1-bp insertion. The blank triangle indicates the CRISPR target site in the fourth exon of ZmACD6. A sequence alignment of the deletion and insertion sites of ZmACD6-1 with the B73 sequence is shown; the protospacer-adjacent-motif (PAM) location is marked in red. (b,c) Maize seedlings were infected with U. maydis wild-type strain SG200, and disease symptoms were scored at 8 days post infection (dpi) (upper panel). The disease rating of plants infected with U. maydis wild-type strain SG200 at 8 dpi (lower panel). P-values were calculated by Student’s t-test (*P < .05; **P < .01); n = number of infected plants. These experiments were repeated three times (b) or twice (c) with similar results.

An inbred maize line with high levels of zmACD6 expression has increased resistance to U. maydis

Since ZmACD6 plays an important role in disease resistance, it can be used to produce hybrids with relatively high disease resistance. Therefore, among a diverse population of natural maize (284 inbred lines), we screened for lines expressing high levels of ZmACD6 transcript using qPCR. ZmACD6 transcript levels were dramatically elevated in the SC-9 line relative to the wild-type B73 line (Figure 6(a)). In Arabidopsis, ACD6 levels were correlated with increased disease resistance.^10,11 In agreement with this finding, when infected with U. maydis SG200, the SC-9 line had less severe symptoms than B73 (Figure 6(b)), suggesting that ZmACD6 positively regulates disease resistance to U. maydis.

Figure 6.

The maize inbred line SC-9 with high ZmACD6 transcript levels has increased resistance to U. maydis and an enhanced defense response. (a) qPCR analysis of ZmACD6 expression in the SC-9 inbred line (b) The disease rating of B73 and SC-9 infected with U. maydis strain SG200 at 8 days post infection (dpi). (c-e) The levels of ZmPR4, ZmPR5, and ZmPR10 transcript in line SC-9, measured by qRT-PCR. P-values were calculated by Student’s t-test (*P < .05; **P< .01;* **p < .01). This experiment was repeated three times (b) or twice (a, c-e), and similar results were obtained.

To test whether the enhanced disease resistance in the SC-9 plants is associated with enhanced defense activation, we tested the expression of three SA-signaling markers (ZmPR4, ZmPR5, and ZmPR10). Compared to wild-type (B73) plants, SC-9 plants showed higher levels of ZmPR4, ZmPR5, and ZmPR10 transcript (Figure 6(c-e)).

Discussion

Maize is an important cereal crop and a model for functional genomics research, but disease causes massive annual yield losses worldwide. Developing crop varieties with broad-spectrum resistance would limit losses due to disease. ACD6 is a key component of broad-spectrum resistance in Arabidopsis and a valuable candidate for the engineering of broad-spectrum resistance in breeding programs.^10,13,14 Here, we reveal that ZmACD6 is the maize ortholog of ACD6. Little is known about the SA-mediated defense network in maize. Our work provides a molecular clue as to the induction of a SA-mediated response in maize. Moreover, from a natural population, we identified a maize line (SC-9) with relatively high ZmACD6 expression levels. Thus, our findings also provide a practical approach for breeding elite varieties with improved resistance.

The ankyrin repeat is a common protein domain in organisms ranging from viruses to humans.²² Ankyrin repeat-containing proteins play important roles in diverse processes, such as transcriptional initiation, cell cycle regulation, cytoskeleton interaction, and transduction.²³ However, only a few ankyrin repeat-containing proteins have been identified and characterized in plants.^18,24,25 ACD6 represents a category of ankyrin repeat-containing proteins with a C-terminal transmembrane domain.²⁴ There are 36 ACD6-like genes in the Arabidopsis genome and 15 ACD6-like genes in the maize genome, but none of them has been assigned a function.^18,24

In this study, we identified ANK23/ZmACD6 as a maize ortholog of ACD6 that complemented the Arabidopsis acd6-2 mutant. Moreover, we identified two ZmACD6 knockout lines with reduced resistance to U. maydis and a line with high levels of ZmACD6 expression (SC-9) with elevated disease resistance to U. maydis. Thus, we have presented multiple lines of evidence that ZmACD6 is critically involved in the response to U. maydis infection. In support of this notion, SC-9 plants have enhanced defense activation. However, it is also possible that the enhanced disease resistance and activation of defense signaling in the SC-9 line are caused by another gene(s) due to the genetic background. Generating transgenic plants that overexpress ZmACD6 would help resolve this question. As ankyrin repeats mediate protein–protein interactions,¹⁶ future studies should aim to identify ZmACD6-interacting proteins.

Materials and methods

Plant materials and growth conditions

The ACD6 loss-of-function mutant acd6-2 was previously described in the Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) ecotype background.¹⁵ Arabidopsis plants were grown in a growth chamber at 20 to 22°C with a 16-h-light/8-h-dark photoperiod. The natural maize (Zea mays) population used in this study was a global collection of 284 inbred lines with abundant genetic variation and were previously described.²⁶ Inbred line KN5585 was used for maize transformation. All maize plants were grown in a growth chamber at 22 to 28°C with a 12-h-light/12-h-dark photoperiod.

Phylogenetic analysis

The ACD6-like sequences were aligned using MEGA 7.0 software,²⁷ and a phylogenetic tree was generated by the neighbor-joining method based on whole protein sequences. One thousand bootstrap replicates were used for statistical support for the nodes in the phylogenetic tree.

Vector construction and Arabidopsis transformation

ZmACD6 full-length cDNA was isolated from maize B73 and confirmed by sequencing. The cDNA was then inserted into the overexpression vector 35S-pGreen via the KpnI and XbaI sites. Flowering acd6-2 plants were transformed with the 35S:ZmACD6 vector using Agrobacterium tumefaciens GV3101 and the floral dip method.²⁸ Independent transgenic progeny (T0) were selected on soil using BASTA treatment. At least five independent transformed lines expressing high levels of ZmACD6 were identified and used for further experiments.

Chemical treatment

For SA treatments, 10-day-old maize seedlings were sprayed with 200 μM SA dissolved in water with 0.005% Silwet-77 until all of the leaves were wet. Leaves were then collected at 0, 6, 12, and 24 h after the treatment.

Pathogen infection

Maize plants were infected with U. maydis SG200 (a kind gift from professor Gunther Doehlemann) using the procedure of Redkar et al. (https://bio-protocol.org/e1760). Briefly, SG200 was grown in YEPSL medium overnight, until the OD₆₀₀ reached 0.6 to 0.8, to ensure that the U. maydis sporidia were in the active dividing phase of growth. SG200 were harvested and washed in sterile water. The cells were then resuspended in water to OD₆₀₀ = 1.0 and 7-day-old maize seedlings were infected with this suspension using a syringe. Eight days later, the morphological symptoms of the infected seedlings were observed and scored. Arabidopsis was infected with Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) and the bacterial growth curve was analyzed as described previously.²⁹

Gene editing

ZmACD6 (GRMZM2G123977) was knocked out using the CRISPR/Cas9 system.³⁰ Guide RNA was designed using CRISPR-P, a web tool for synthetic single-guide RNA design in plants (http://crispr.hzau.edu.cn/CRISPR2/). The guide RNA sequence is 5′-GCTAGGCCGCCATTTCCAGT-3′. The guide RNA was inserted into the pBUE411 vector. Confirmed clones were transferred to Agrobacterium EHA105, and T0 transgenic plants were identified by both the bialaphos (bar) strip test and sequencing of ZmACD6. Eight T0 transgenic lines that tested positive in the bar strip test and harbored mutations in the target region were chosen and self-pollinated or backcrossed to KN5585 to generate T1. Four T1 transgenic lines with different mutations in the target region were planted in the field. Each plant was genotyped by targeted sequencing, and the heterozygous and homozygous mutants were self-pollinated. The homozygous lines with editing mutations were used in further experiments.

qPCR analyses

Total RNA preparations were isolated from seedlings using TRIzol reagent (Takara) according to the manufacturer’s protocol. The first-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real-time, Takara) according to the manufacturer’s protocol. TB Green Premix Ex Taq II (Takara) and the LightCycler 96 (Roche) PCR system were used for qPCR. qPCR was performed as described previously.³¹ The primer sequences employed to amplify the target genes are listed in Supplementary Table S1. The amplification efficiency for each primer pair was calculated using eight 3-fold cDNA dilutions (Supplementary Figure S3). Melting curves were generated for each reaction to ensure amplification specificity (Supplementary Figure S4). The comparative CT method was used to calculate the expression level,³² and the maize ACTIN gene was used as the reference.

Funding Statement

This work was supported by the startup funds from Agricultural University of Hebei [ZD201609];The national natural science foundation of China [31601381];the Science and Technology Research Key Project of Colleges and University in Hebei Province [ZD2017037].

Acknowledgments

We express our sincere gratitude to Gunther Doehlemann, Xiaodong Wang, and Yuanyuan Yan for sharing reagents and strains. This work was supported by grants to ZZ from The National Natural Science Foundation of China (31601381) and startup funds from Agricultural University of Hebei (ZD201609). JG was supported by the Science and Technology Research Key Project of Colleges and University in Hebei Province (ZD2017037).

Author contributions

ZZ designed the research. ZZ, JG, and YZ performed the experiments and analyzed the data. ZZ wrote the article. JC supervised the experiments.

Conflict of Interest Statement

The authors declare that they have no conflict of interests.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website