
==== Front
Plant Physiol
Plant Physiol
plphys
Plant Physiology
0032-0889
1532-2548
Oxford University Press US

38753366
10.1093/plphys/kiae286
kiae286
Research Article
Signaling and Response
AcademicSubjects/SCI01270
AcademicSubjects/SCI01280
AcademicSubjects/SCI02286
AcademicSubjects/SCI02287
AcademicSubjects/SCI02288
Effector Cs02526 from Ciboria shiraiana induces cell death and modulates plant immunity
https://orcid.org/0009-0008-4831-8313
Zhang Shuai State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0009-0005-4103-0825
Li Ruolan State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0000-0003-3501-8860
Fan Wei State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0009-0002-1054-7299
Chen Xuefei State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0009-0009-0659-5897
Liu Shuman State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0009-0003-2356-196X
Zhu Panpan Resource Institute for Chinese & Ethnic Materia Medica, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

https://orcid.org/0009-0007-4597-7906
Gu Xiaohui State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

https://orcid.org/0009-0007-0373-5829
Wang Shuchang Chinese Academy of Tropical Agricultural Sciences, Institute of Environment and Plant Protection, Haikou 570100, China

https://orcid.org/0000-0001-6140-0102
Zhao Aichun State Key Laboratory of Resource Insects, Institute of Sericulture and Systems Biology, Southwest University, Chongqing 400715, China

Author for correspondence: zhaoaichun@hotmail.com; zhaoaichun@swu.edu.cn
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is Aichun Zhao.

Conflict of interest statement. None declared.

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© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.
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Abstract

Sclerotinia disease is one of the most devastating fungal diseases worldwide, as it reduces the yields of many economically important crops. Pathogen-secreted effectors play crucial roles in infection processes. However, key effectors of Ciboria shiraiana, the pathogen primarily responsible for sclerotinia disease in mulberry (Morus spp.), remain poorly understood. In this study, we identified and functionally characterized the effector Cs02526 in C. shiraiana and found that Cs02526 could induce cell death in a variety of plants. Moreover, Cs02526-induced cell death was mediated by the central immune regulator brassinosteroid insensitive 1-associated receptor kinase 1, dependent on a 67-amino acid fragment. Notably, Cs02526 homologs were widely distributed in hemibiotrophic and necrotrophic phytopathogenic fungi, but the homologs failed to induce cell death in plants. Pretreatment of plants with recombinant Cs02526 protein enhanced resistance against both C. shiraiana and Sclerotinia sclerotiorum. Furthermore, the pathogenicity of C. shiraiana was diminished upon spraying plants with synthetic dsRNA-Cs02526. In conclusion, our findings highlight the cell death-inducing effector Cs02526 as a potential target for future biological control strategies against plant diseases.

An effector in Ciboria shiraiana induces cell death, and treatment with its double-stranded RNA or protein improves plant resistance to pathogens.

Hainan Province Science and Technology ZDYF2022SHFZ319 China Agriculture Research System CARS-18-ZJ0201 Chongqing Modern Agricultural Industry Technology COMAITS202311
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pmcIntroduction

Plants are often challenged by various microbes during their growth and development. During long-term co-evolution, dynamic changes occur between plant immunity and microbe pathogenicity (Chisholm et al. 2006). Plant immune systems mainly respond to pathogens in 2 ways: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (Jones and Dangl 2006).

PAMPs are evolutionarily conserved molecules commonly found in bacteria, oomycetes, and fungi that play essential roles in microbial survival (Thomma et al. 2011). PAMPs found in bacteria mainly include flagellin, elongation factors, peptidoglycans, lipopolysaccharides (LPS), and cold shock proteins. Among them, flg22 and elf18 are the best characterized (Zipfel 2009). PAMPs such as chitin, glycoside hydrolase family 12 (GH12), and Valsa mali VmE02 in fungi have also been well studied (Shinya et al. 2015; Nie et al. 2019; Zhang et al. 2021a). Moreover, large numbers of PAMPs have been reported in oomycetes, including elicitin infestin1 INF1 GH12 protein XEG1, expansin-like proteins EXLX1, and aldose 1-epimerase AEP1 (Du et al. 2015; Xu et al. 2021; Pi et al. 2022). However, the function of PAMPs in the fungi of the Sclerotiniaceae family and the mechanisms underlying their effects on plants are less studied.

The recognition of PAMPs by plant plasma membrane-localized pattern recognition receptors (PRRs) leads to the activation of PTI in plants (Monaghan and Zipfel 2012). Hundreds of potential PRRs are encoded in plant genomes, including receptor-like kinases (RLKs) and receptor-like proteins (RLPs). RLKs have extracellular, transmembrane, and intracellular kinase domains, whereas RLPs lack the intracellular domain (Albert et al. 2020). BRI1-associated kinase-1 (BAK1)/somatic embryogenesis receptor kinase 3 and suppressor of BIR1-1 (SOBIR1) play crucial roles in regulating PRR-mediated plant immunity (Liebrand et al. 2014). BAK1 usually acts as a co-receptor by forming complexes with PRRs and is pivotal to immune signaling induced by PAMP recognition. For example, in Arabidopsis (Arabidopsis thaliana), RLK flagellin-sensing-2 (FLS2) recruits BAK1 to form the receptor complex that recognizes the flagellin epitope flg22 (Chinchilla et al. 2007). In contrast, SOBIR1 usually serves as a common adaptor and associates with diverse RLPs to form RLP adaptor complexes, after which downstream defense responses are activated. For example, in tomato (Solanum lycopersicum), BAK1 and SOBIR1 interact with cf4 to form a complex that is essential for defense against Avr4 secreted by Cladosporium fulvum (Liebrand et al. 2013). Furthermore, BAK1 and SOBIR1 can associate with RE02, an RLP receptor for VmE02 (a PAMP from V. mali), to form a tripartite complex that induces plant resistance in Nicotiana benthamiana (Nie et al. 2021).

RNA interference (RNAi) is a conserved mechanism that regulates gene expression in eukaryotic organisms and has been widely used in function characterization (Li et al. 2010). Based on RNAi, multiple control strategies have been conducted in plants, such as host- or spray-induced gene silencing (HIGS or SIGS, respectively) (Koch et al. 2019). For instance, via spray application, 3 genes from the ergosterol biosynthesis pathway were found to be involved in the growth and pathogenicity of Botrytis cinerea (Duanis-Assaf et al. 2022). Additionally, the downregulated expression of a nematode chitin synthase gene via HIGS was found to enhance soybean resistance to both Heterodera glycines and Fusarium oxysporum (Kong et al. 2022). Compared with HIGS, SIGS could prevent the production of genetically modified organisms. Therefore, the SIGS application holds potential for developing an environmentally friendly approach to control plant diseases.

Mulberry (Morus spp.) is an economical tree that plays an important role in the sericulture industry. Furthermore, there are rich nutrients, anthocyanin, and other substances in mulberry fruits that are responsible for their high value and broad research appeal (Li et al. 2020). Ciboria shiraiana, a necrotrophic pathogen from the Sclerotiniaceae family, is the main causal agent of mulberry sclerotinia disease, which causes severe losses to annual mulberry fruit yield (Zhu et al. 2021; Zhang et al. 2021b). Necrotrophic pathogens can kill plant cells and absorb their nutrients. Effector-induced host cell death is central to the infection mechanisms of pathogens (McDonald and Solomon 2018). Therefore, a comprehensive understanding of the effector function can help the development of new strategies to control plant diseases (Dagvadorj et al. 2022). However, to our knowledge, no studies have focused on effectors in C. shiraiana.

MD-2-related lipid recognition (ML) superfamily proteins, including MD-1, MD-2, Niemann–Pick-type C2 (Npc2) protein, GM2 activator protein, phosphatidylglycerol/phosphatidylinositol transfer proteins (PG/PI-TP), and mite allergen Der p 2, bind to specific lipids and play important roles in lipid recognition and metabolism in mammals (Liao et al. 2011). However, the functions of the ML protein in phytopathogenic fungi remain relatively unexplored. In this study, we identified an ML family protein, Cs02526, secreted by C. shiraiana that can trigger plant cell death and analyzed its function in plant immunity and pathogenicity. Overall, our findings highlighted Cs02526 as a potential target for disease control and provided an experimental basis for future studies concerning C. shiraiana–plant interactions.

Results

Cs02526 was an apoplastic protein with an ML domain that induces cell death in N. benthamiana

To investigate the functions of effectors in C. shiraiana, potential effector genes were screened in the C. shiraiana genome. Transient expression experiments were conducted for 47 successfully cloned effectors in N. benthamiana (Supplementary Fig. S1, A and B). The results showed that Cs02526, containing an ML domain (cl00274), induced cell death in N. benthamiana (Fig. 1A). BAX was used as a positive control, while green fluorescent protein (GFP) was the negative control. Western blot analysis indicated that all proteins were expressed in N. benthamiana (Fig. 1B). In addition, electrolyte leakage and expression levels of 2 hypersensitive response (HR)-specific marker genes, NbHSR203J and NbHIN1, were analyzed in N. benthamiana agroinfiltrated with the Cs02526 construct. The results showed that the electrolyte leakage and expression levels of target genes in agroinfiltrated leaves were significantly increased compared with those in the control group (Fig. 1, C and D). These results demonstrated that Cs02526 could induce cell death in N. benthamiana.

Figure 1. Cs02526 triggered cell death in N. benthamiana.A) The cell death-inducing abilities of Cs02526 and Cs02526ΔSP were determined in N. benthamiana. In the schematic diagram of the Cs02526 protein structure (above), related information were labeled on the protein fragments. Cell death was induced by different constructs (below left) at 7 d after agroinfiltration (dpa) and decolorization diagram (below right). B) Western blot analysis of proteins expressing GFP, Cs02526, and Cs02526ΔSP fused with a GFP tag. Coomassie Brilliant Blue (CBB) staining was used to indicate equal loading in each sample. C) Quantification of cell death by measuring the electrolyte leakage in N. benthamiana leaves at 7 dpa. Six leaf discs (9 mm diameter) were measured in each group. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, **P < 0.01; ***P < 0.001. D) Relative expression levels of HR-specific marker genes in N. benthamiana. Three leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student's t test, **P < 0.01; ***P < 0.001.

The protein encoded by Cs02526 had 86 amino acids and an N-terminal signal peptide (SP, 1 to 19 amino acids) (Fig. 1A). To determine whether the SP was important for Cs02526-induced cell death, Agrobacterium tumefaciens carrying a PVX: Cs02526ΔSP vector was agroinfiltrated into N. benthamiana leaves. As shown in Fig. 1A, compared with Cs02526, Cs02526ΔSP failed to induce cell death, and electrolyte leakage and hypersensitive-related gene expression levels were significantly decreased (Fig. 1, B and C). These results indicated that the SP is crucial for Cs02526-induced cell death.

Cs02526 was predicted to be an apoplastic effector using EffectorP (Supplementary Table 1). To verify this prediction, the subcellular localization of Cs02526 in N. benthamiana was investigated. Cs02526 was found to be localized to the cell edge (Supplementary Fig. S2A). To further distinguish the GFP signals from the apoplast and plasma membrane, N. benthamiana leaves were plasmolyzed with 30% sucrose. As shown in Supplementary Fig. S2B, Cs02526 but not Cs02526ΔSP can be observed in the apoplast of N. benthamiana cells after plasmolysis. These results confirmed Cs02526 as an apoplastic effector. Overall, the above data suggested that Cs02526 must be targeted to the extracellular space of N. benthamiana tissue to induce cell death.

Cs02526 triggered immune responses in N. benthamiana and mulberry

To determine whether Cs02526-induced cell death was accompanied by plant defense responses, reactive oxygen species (ROS) accumulation and callose deposition were analyzed in N. benthamiana leaves agroinfiltrated with the Cs02526 construct. Compared with those in the control group, substantial ROS accumulation and callose deposition were observed in N. benthamiana leaves agroinfiltrated with the Cs02526 construct (Fig. 2, A and B). Cs02526 was localized in the plant apoplast, so it was speculated that it may have a function similar to that of PAMPs. To verify this assumption, the expression levels of PTI-related genes were determined using RT-qPCR. The results showed that the expression of PTI marker genes, including NbCYP71D20, NbPTI5, NbACRE31, NbWRKY7, and NbWRKY8, were markedly increased in the Cs02526 group (Fig. 2C). The expression levels of defense marker genes involved in the plant hormone signaling pathway, such as NbPR1a, NbPR2, NbPR4, NbLOX, and NbERF1, which are marker genes of salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene (ET)-dependent immunity, were also validated. Compared with those of the control group, the expression levels of these genes were significantly upregulated in the leaves agroinfiltrated with the Cs02526 construct (Fig. 2C). These results indicated that Cs02526 induced plant PTI and activated defense pathways mediated by SA, JA, and ET. Similarly, compared with those in the control group (empty vector protein), the expression levels of immune response-related genes in mulberry leaves were considerably upregulated in the Cs02526 group (Supplementary Fig. S3D). These findings revealed that Cs02526 could trigger immune responses in plants.

Figure 2. Plant immunity responses triggered by Cs02526 were dependent on BAK1, but not SOBIR1 receptors in plants. A) Accumulation of ROS and B) deposition of callose were determined in N. benthamiana. The scale bar in B) represents 200 μm. C) The relative expression levels of PAMP-triggered immunity (PTI) marker genes and hormone-related defense genes were detected in N. benthamiana leaves induced by Cs02526 construct for 2 dpa. Three leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001. D) Cell death triggered by BAX, Cs02526 in tobacco rattle virus (TRV): GFP, TRV: BAK1, and TRV: SOBIR1-treated N. benthamiana leaves. Cell death was photographed at 7 dpa. E) Target gene expression levels were determined by RT-qPCR analysis in silenced lines. Three leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, **P < 0.01; ***P < 0.001. F) The relative expression level of NbCYP71D20 was detected in silenced lines agroinfiltrated with the Cs02526 construct for 36 h. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, **P < 0.01.

To further validate the ability of Cs02526 to induce plant cell death, Cs02526 was expressed in Escherichia coli (Supplementary Fig. S3A), and various concentrations of purified protein ranging from 0.1 to 200 μg/mL were infiltrated into N. benthamiana and mulberry leaves using a needleless syringe. The recombinant Cs02526 protein induced cell death at a concentration of 100 μg/mL in N. benthamiana and mulberry leaves (Supplementary Fig. S3C). To further analyze whether Cs02526 could induce cell death in other plants, the purified protein of Cs02526 was injected into the leaves. Cs02526 was found to induce cell death in N. benthamiana, tobacco (Nicotiana tabacum), tomato (S. lycopersicum), strawberry (Fragaria vesca), and mulberry (Morus alba) leaves, but cell death was not observed in the control group (Supplementary Fig. S3B). These results demonstrated that Cs02526 could induce cell death in various plant species.

Cs02526-induced cell death depended on BAK1 but not SOBIR1

PAMPs are recognized by PRRs in plants, which induced immune responses. Among all PRRs, the co-receptors BAK1 and SOBIR1 play important roles. To investigate whether cell death induced by Cs02526 was dependent on BAK1 and SOBIR1, virus-induced gene silencing (VIGS) was adopted to silence NbBAK1 and NbSOBIR1 in N. benthamiana (Supplementary Fig. S4A). The results showed that cell death induced by Cs02526 was abolished in the BAK1-silenced lines, but was not affected in the SOBIR1-silenced and control lines (Fig. 2D). In contrast, as positive control, BAX induced cell death in all lines. Western blot analysis showed that the proteins were expressed in all lines (Supplementary Fig. S4B). According to RT-qPCR results, the expression levels of NbBAK1 and NbSOBIR1 were significantly reduced in corresponding plants (Fig. 2E). In Cs02526-agroinfiltrated leaves, the relative expression level of the PTI marker gene NbCYP71D20 in BAK1-silenced plants was decreased to about 30% (Fig. 2F). These results showed that the co-receptor BAK1 was required for Cs02526-triggered immune responses.

Cs02526 homologs were widely distributed in hemibiotrophic and necrotrophic phytopathogenic fungi, but its function was not conserved

To investigate whether Cs02526 homologs were present in other species, the phylogenetic distribution of Cs02526 was analyzed in different organisms. Utilizing the Cs02526 protein sequence, a BLAST analysis was conducted across oomycete, fungal, bacterial, and plant species. The results showed that homologs were only in fungi (Fig. 3A). Notably, Cs02526 homologs were distributed in hemibiotrophic and necrotrophic pathogens, but not in biotrophic pathogens (Fig. 3A). Phylogenetic analysis revealed that the evolutionary distance between Cs02526 and SS1G_07613 (XP_001590989.1) from Sclerotinia sclerotiorum was the closest (Fig. 3B). To determine whether other homologs would induce cell death, we selected homologous proteins with high similarity from 5 phytopathogenic fungi for analysis. Transient expression experiments showed that these homologs could not induce obvious cell death in N. benthamiana leaves (Fig. 3C). In conclusion, Cs02526 homologs were widely distributed in phytopathogenic fungi, but their functions may have undergone divergence.

Figure 3. Cs02526 homologs were widely distributed across fungal species but could not induce cell death. A) Number of Cs02526 homologs in different species. B) Phylogenetic analysis of Cs02526 and its homologs from 22 fungal species. Different evolutionary branches were marked with different background colors and the corresponding lifestyles were labeled behind. Bootstrap percentage support for each branch is indicated. The scale bar indicates the branch length. C) Cell death responses induced by different Cs02526 homologs at 7 dpa were analyzed in N. benthamiana with SS1G_07613 from S. sclerotiorum, Bcnpc2 from B. cinerea, FGSG_09586 from F. graminearum, MGG_01557 from Magnaporthe oryzae, and VDAG_00559 from V. dahliae.

A 67-amino acid fragment of Cs02526 was required to induce cell death

To identify how Cs02526 induces cell death, we investigated whether immunogenic fragments in Cs02526 were critical for inducing activity. N- and C-terminal truncated mutants were generated and agroinfiltrated into N. benthamiana leaves. All truncated mutants (N1/2/3, C1/2/3) failed to induce cell death (Fig. 4A). Cell death was observed in the heat-treated group, indicating that the purified Cs02526 protein (67 aa) can induce plant cell death (Fig. 4B). These findings suggested that a 67-amino acid fragment, rather than a tertiary structure, was required for Cs02526-induced cell death. In addition, why Cs02526 homologs failed to induce plant cell death was explored. Cs02526 exhibited differences in the C-terminal compared with its homologs, with C-terminal extensions observed in other homologs (Fig. 4C). These results indicated that Cs02526 and its homologs encode different functional proteins, which is consistent with their 3-dimensional structure (Fig. 4C and Supplementary Fig. S5). To investigate whether C-terminal differences affect the homolog's ability to induce cell death, we replaced the C-terminus of the homologs with that of Cs02526. The results showed that, except for SS1G_07613-CM, which restored the ability to induce cell death, the other mutants remained unable to induce cell death (Fig. 4D). The main reason is that SS1G_07613-CM protein sequence is highly similar to that of Cs02526. The above results indicated that a 67-amino acid fragment of Cs02526 was required to induce cell death.

Figure 4. A 67-amino acid fragment of Cs02526 was required to induce cell death in n. benthamiana.A) Regions of Cs02526 were examined for the cell death activity by the agrobacterium-mediated transient expression. Schematic presentation of the Cs02526 mutants. Cell death was observed at 7 dpa. Related information were labeled on the protein fragments. B) Cell death responses at 5 dpa induced by heat-treated Cs02526 and emptor vector (EV) protein in N. benthamiana leaves. The protein samples were boiled for 10 min and left to stand at RT before injection. C) Alignment of the sequences of Cs02526 and homologs from other fungi. The black and blue lines represent the ML domain and the phosphatidylglycerol/phosphatidylinositol (PG/PI) transfer protein, respectively. The red box indicates the amino acid residues at the C-terminus that are different from the homologous sequence of Cs02526. D) Various mutants were constructed and transiently expressed in 4-wk-old N. benthamiana leaves. Photographs were taken 7 dpa. CM stands for C-terminal mutant.

Cs02526 was involved in hyphal radial growth and osmotic stress responses in C. shiraiana

RNAi was used to investigate the biological function of Cs02526 in C. shiraiana. Supplementary Figure S6C showed that the relative expression levels of Cs02526 were downregulated to 40% to 50% in silenced strains. The growth and development of wild-type (WT) and silenced strains were observed on potato dextrose agar (PDA) medium. After 24 and 48 h of cultivation, the hyphal growth of Cs02526-silenced strains was faster than that of the WT strains. However, there was no significant difference in the number and weight of sclerotia at 14 d (Supplementary Fig. S6, A, B, and D). Next, whether Cs02526 was involved in stress responses was investigated. The hyphal radial growth rate of the WT and silenced strains on PDA containing the osmotic stress agents NaCl and KCl, oxidative stress agent H2O2, and cell wall-disturbing agent SDS was measured. As shown in Supplementary Fig. S7A, there was no significant difference in the growth and inhibition rates of hyphal growth between WT and silenced strains in PDA containing H2O2 and SDS. However, in the medium containing osmotic stress agents (NaCl and KCl), the growth of hyphae in silenced strains was suppressed, and the inhibition rate of hyphal growth was approximately 70% and 45%, respectively (Supplementary Fig. S7, A and B). These results demonstrated that Cs02526 was involved in hyphal radial growth and osmotic stress responses.

Cs02526 was a virulence-related gene and topical application of dsRNA-Cs02526 inhibited the virulence of C. shiraiana

To investigate whether Cs02526 contributes to the virulence of C. shiraiana, RT-qPCR analysis was conducted to determine the expression patterns of Cs02526. As shown in Supplementary Fig. S8, the relative expression level of Cs02526 was increased after inoculation in leaves and peaked at 12 h postinfection (hpi). This finding implied that Cs02526 might be involved in the infection process. Then, detached leaves were inoculated with mycelial plugs. Compared with those in the WT strains, lesions and pathogen biomass were reduced in silenced strains. These results indicated that Cs02526 was required for pathogenicity in C. shiraiana (Fig. 5, A, C, and D). Moreover, to evaluate the potential of Cs02526 as a virulence factor in biological control, the SIGS method was used. First, the efficiency of dsRNA uptake in C. shiraiana was evaluated by treating fungal cells with Cy3-labeled dsRNA. Fluorescence was observed at the hyphal tip of C. shiraiana after 18 h of treatment (Supplementary Fig. S9). This finding suggested that C. shiraiana could uptake dsRNA from the environment. Next, dsRNA-Cs02526 was synthesized and used in follow-up experiments, with dsRNA-GFP as a negative control. In the dsRNA-Cs02526 group, the lesions in N. benthamiana and mulberry leaves were significantly smaller than those in the control group (Fig. 5B). Furthermore, RT-qPCR analysis showed that the expression levels of Cs02526 were considerably reduced in the dsRNA-Cs02526 group (Fig. 5, E and F). In conclusion, Cs02526 was essential for the pathogenicity of C. shiraiana and has potential as an RNAi target to control plant diseases.

Figure 5. Cs02526 could be a target for disease control. A) Lesions of N. benthamiana (36 hpi) and mulberry (48 hpi) leaves caused by the WT and Cs02526-silenced strains. B) Representative pictures of N. benthamiana and mulberry leaves treated with dsRNA-GFP and dsRNA-Cs02526 following spray inoculation with C. shiraiana were taken at 36 or 48 hpi. C) Lesion diameters of leaves were measured at 36 or 48 hpi. Lesion diameters were assessed over 3 independent experiments (n = 9). Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, ***P < 0.001. D) The biomass of C. shiraiana in N. benthamiana was determined by RT-qPCR. Three leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, ***P < 0.001. E) The relative lesion sizes were measured at 36 hpi. Lesion areas were assessed from 3 independent experiments (n = 15). Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, ***P < 0.001. F) The expression level of Cs02526 was detected by RT-qPCR analysis. Five leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, ***P < 0.001.

Cs02526 enhanced plant disease resistance to C. shiraiana and S. sclerotiorum

Next, we confirmed whether Cs02526 could enhance plant disease resistance. Figure 6A and B shows that after treatment with 50 μg/mL recombinant Cs02526 protein, the lesion areas in N. benthamiana leaves were significantly reduced compared with those in the control group. RT-qPCR confirmed that the C. shiraiana biomass was decreased compared with EV control (Fig. 6C). Furthermore, disease resistance to S. sclerotiorum in N. benthamiana leaves was investigated. Compared with those in the control group, the lesions and fungal biomass were significantly reduced in the Cs02526-treatment group (Fig. 6, D to F). Taken together, these results indicated that Cs02526 could enhance plant resistance to diverse pathogens from the Sclerotiniaceae family.

Figure 6. Cs02526 enhanced plant resistance to C. shiraiana and S. sclerotiorum.A and D) Phenotypes of N. benthamiana leaves inoculated with C. shiraiana and S. sclerotiorum at 24 hpi under UV light. The leaves were pretreated with purified Cs02526 (50 μg/mL). EV protein was used as a control. B and E) Lesion areas on pretreated leaves were measured at 24 hpi. Lesion areas were assessed from 3 independent experiments (n = 9). Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, *P < 0.05; ***P < 0.001. C and F) The fungal biomass of C. shiraiana and S. sclerotiorum in N. benthamiana was determined by RT-qPCR. Three leaves were counted in each replicate. The experiment was repeated 3 times. Data were represented as the mean ± Sem. The asterisks indicate significant differences by Student’s t test, ***P < 0.001.

Discussion

Cs02526 was a cell death-inducing effector in C. shiraiana

Effectors secreted by pathogens play crucial roles in plant–pathogen interactions (Giraldo and Valent 2013). Genomic and transcriptomic data from Fusarium graminearum and Phytophthora sojae have been used to identify many effector proteins involved in pathogenic infection (Wang et al. 2011; Lu and Edwards 2016). Identifying virulence-related effectors and elucidating their roles are necessary to understand the pathogenic mechanisms. In the present study, a bioinformatics analysis of genomic data was conducted, focusing on the selection of proteins with features such as SPs and the absence of transmembrane domains. We finally obtained 72 potential effector proteins (Supplementary Fig. S1A). Then, 47 candidates were transiently expressed in N. benthamiana. Among them, the unique effector protein Cs02526 could induce cell death in plants (Supplementary Fig. S1B).

Cs02526 homologs were widely distributed in hemibiotrophic and necrotrophic pathogens but were absent in biotrophic pathogens. According to our findings, Cs02526 homologs did not induce cell death in N. benthamiana (Fig. 3C). A possible explanation for this phenomenon was that hemibiotrophic and necrotrophic pathogens could uptake nutrients from dead cells, while biotrophic pathogens could not (Oliver and Ipcho 2004). Cell death, as an HR, blocked the infection of biotrophic pathogens. Therefore, during the evolutionary process, biotrophic and hemibiotrophic pathogens likely discarded genes or modified the functions of these genes that induced plant cell death, enabling them to successfully colonize host plants. In our study, only Cs02526, secreted by C. shiraiana, was found to induce cell death, which might be due to the different evolutionary pathways of different pathogens (Raffaele and Kamoun 2012). Another explanation may be that these proteins could have different functions. Multiple sequence alignment indicated that, compared with its homologs, the Cs02526 protein has a shorter protein sequence. The Cs02526 protein has an ML domain, while others are PG/PI-TP (Fig. 4C and Supplementary Fig. S5). The MD2 protein mediates the transmission of LPS signaling pathways through interactions with LPS ligands, which are PAMPs that trigger the innate immune response in mammals (Inohara and Nuñez 2002). PG/PI-TP is a PG/PI exchange protein that is crucial for cellular vitality and membrane transport (Cockcroft and Garner 2011). Furthermore, we demonstrated that a 67-amino acid fragment was important for Cs02526-induced cell death (Fig. 4, Supplementary Table S2). The C-terminal extension in the homologs might result in insufficient immune-triggering fragments. Furthermore, there may be no corresponding receptor in N. benthamiana to recognize the homologs and trigger cell death. The above explanations were supported by the results shown in Figs. 3 and 4.

Cs02526-induced plant cell death depends on the co-receptor BAK1

The apoplastic space is a complex battleground for plant–microbe interactions (Wang et al. 2020). In the present study, Cs02526 induced plant cell death in the presence of the SP, but the truncated SP did not induce cell death (Fig. 1A). Meanwhile, bioinformatics analysis and subcellular localization assay indicated that Cs02526 was an apoplastic effector (Supplementary Table S1 and Fig. S2). These results suggested that the apoplastic space was critical for Cs02526-induced cell death. Similarly, the FoEG1 and AEP1 proteins from F. oxysporum and P. sojae, respectively, also required SP to induce cell death (Xu et al. 2021; Zhang et al. 2021a). These proteins elicited plant immune responses as PAMP molecules, and Cs02526 was shown to perform the same function.

Plants recognize pathogen invasion in the apoplastic space through PRRs. Among them, the co-receptors BAK1 and SOBIR1 play important roles in pathogen recognition and immune signaling as RLKs (Liang and Zhou 2018). Therefore, their involvement is required for the activation of immune responses or induction of cell death by many apoplastic effectors. For example, Verticillium dahliae secretes the GH12 protein VdEG1, which induces cell death and immune responses depending on the BAK1 and SOBIR1 co-receptors in N. benthamiana (Gui et al. 2017). Additionally, Rhynchosporium commune RcCDI1-induced cell death was BAK1 and SOBIR1 dependent (Franco-Orozco et al. 2017). In contrast, Cs02526-induced cell death in N. benthamiana requires BAK1 but not SOBIR1 (Fig. 2D). Likewise, cell death induced by the oomycete apoplastic protein PcEXLX1 depends on BAK1 but not SOBIR1 (Pi et al. 2022). The above results indicated that although all of these effector function as PAMPs, they induce plant immunity in different ways. Plant-derived membrane receptors, such as FLS2 and RE02, recognize corresponding PAMPs (Chinchilla et al. 2007; Nie et al. 2021). For example, FLS2, a leucine-rich repeat (LRR)-RLK identified in A. thaliana, can specifically recognize the flagellin epitope flg22 (Chinchilla et al. 2007). Owing to the dependence of Cs02526-induced cell death in N. benthamiana on the co-receptor BAK1, we speculated that the corresponding receptor for Cs02526 in plants might form an LRR-RLKs or LRR-RLPs/BAK1 complex. This complex may enable Cs02526 to trigger signal transduction in cells and activate downstream immune responses. However, this speculation needs to be confirmed in future experiments.

Cs02526 showed promise as a target for disease control

When exploring the biological function of Cs02526, we found that Cs02526 expression was upregulated during the early stages of infection (Supplementary Fig. S8). Functional studies using RNAi methods showed that Cs02526 was involved in pathogenicity and osmotic stress responses in C. shiraiana (Fig. 5A; Supplementary Fig. S7A). Cs02526 belongs to the ML protein family, and the main function of this family is to participate in lipid metabolic processes. Cs02526 may contribute to increased synthesis of osmoprotectants, such as glycerol (Zulfiqar et al. 2019). This contributes to balancing the osmotic pressure inside and outside the cell and may induce the expression of genes related to antioxidant defense pathways, enhancing the resistance of fungi to oxidative stress and ensuring cellular homeostasis under osmotic stress (Liu et al. 2021). Cs02526 exhibits dual functions in plant–pathogen interactions. On the one hand, Cs02526 promotes the occurrence of sclerotinia disease, and on the other hand, it activates innate immune response in plants. This seemingly paradoxical phenomenon is actually not untraceable. Over the past few decades, with research on phytopathogens, several effectors have been well studied. As key virulence determinants, these effectors can kill host plant tissue and promote host infection while at the same time inducing plant immune responses. For example, a GH12 protein, XEG1, secreted by P. sojae induces cell death, promotes pathogen colonization, and triggers plant innate immune responses (Ma et al. 2015). Necrosis and ET-inducing peptide 1-like proteins can be secreted by a wide range of plant-associated microorganisms, which can induce cell death and activate bursts of ROS, callose deposition, and so on (Lian et al. 2022; Pirc et al. 2023). With further research into disease-susceptible genes, the Arabidopsis malectin-like domain-containing RLK ANXUR (ANX1 and ANX2) was found to negatively regulate plant disease resistance (Mang et al. 2017). This finding potentially explains the dual function of Cs02526. It raises the possibility of a PRR protein with its encoding gene coinciding with disease susceptibility genes. The precise molecular mechanism underlying this process deserves further investigation.

C. shiraiana is the causal agent of mulberry sclerotinia disease, which causes considerable yield losses annually in mulberry-growing countries (Jiao et al. 2020; Zhang et al. 2021b). However, it has been difficult to develop sclerotinia disease-resistant varieties given the complex resistance mechanisms in plants and the limitations of transgenic technology in mulberry plants. In addition, the overuse of traditional fungicides has contributed to environmental pollution and pathogen resistance. Thus, environmentally friendly methods are urgently required. Key pathogenic factors in microbes have been identified as environmentally friendly biological reagents that could enhance plant disease resistance or reduce pathogenicity (Song et al. 2021; Duanis-Assaf et al. 2022). In our study, treatment with exogenous recombinant Cs02526 protein contributed to plant resistance against C. shiraiana and S. sclerotiorum in N. benthamiana leaves (Fig. 6, A and D), indicating that the application of recombinant Cs02526 protein might be effective at improving the resistance of plants to sclerotinia disease. Meanwhile, spraying dsRNA-Cs02526 on N. benthamiana and mulberry leaves reduced the pathogenicity of C. shiraiana (Fig. 5B). These results suggested that the Cs02526 gene could be used as an environmentally friendly biological agent for sclerotinia disease control. Nanocarriers containing dsRNA-Cs02526 or Cs02526 protein could enhance stability and delivery efficiency into plant cells (Kandhol et al. 2023; Qiao et al. 2023). This biological method would reduce pesticide use and the need for lengthy, costly processes to obtain transgenic plants, especially for many plants with immature transgenic technology.

In conclusion, our findings indicated that C. shiraiana secretes the apoplastic effector Cs02526, which induced strong immune responses in plants (cell death) but also improved pathogenicity, facilitating C. shiraiana infection (Fig. 7A). Additionally, treatment with exogenous low concentrations of Cs02526 protein was found to induce plant immunity and disease resistance (Fig. 7B). Spraying of dsRNA-Cs02526 reduced the transcript level of Cs02526, which contributed to weak pathogenicity (Fig. 7C). These results revealed the roles of Cs02526 as a plant immune inducer and a key virulence factor and indicated that it is an attractive candidate target for controlling plant diseases. Furthermore, Cs02526 has the potential to develop novel biological fungicides.

Figure 7. Proposed model illustrating the roles of Cs02526 in C. shiraiana infection and its potential for disease control. A) The apoplastic effector Cs02526 was secreted by C. shiraiana to elicit potent immunity in plants and induce plant cell death, thereby facilitating infection. B) Treatment with exogenous low concentrations of Cs02526 was found to induce plant immunity, resulting in enhanced plant resistance against pathogens. C) Spraying specific dsRNA on plants downregulated transcript levels of Cs02526, which weakened the pathogenicity of C. shiraiana. RISC represents the RNA-induced silencing complex.

Materials and methods

Strains culture and plant growth conditions

C. shiraiana strain WCCQ01 and S. sclerotiorum strain 1980 were cultivated and maintained on PDA at 25°C. E. coli strains Trans1-T1 and BL21 (DE3) were cultured on LB medium at 37 °C. The A. tumefaciens GV3101 strain was cultured on LB medium at 28°C. N. benthamiana and tobacco (N. tabacum), tomato (S. lycopersicum), strawberry (F. vesca), and mulberry (M. alba cv. ‘Guiyou No. 62’) were grown in a climate chamber at 20 to 25°C with a 16 h photoperiod and 60% relative humidity.

RNA and DNA extraction and RT-qPCR

The collected plant leaves and fungal samples were embedded in liquid nitrogen for RNA or DNA extraction. Genomic DNA and total RNA were extracted using the genomic DNA Kit (TIANGEN Biotech, Beijing, China) and EZ-10 Total RNA Mini-Preps Kit (Sangon Biotech, Shanghai, China) following the manufacturer's instructions. Total RNA (1 μg) was used as the template to synthesize cDNA using the PrimeScript RT Reagent Kit (Takara, Tokyo, Japan). RT-qPCR was conducted using the PrimeScript RT Reagent Kit (Takara). β-actin genes were used as internal controls. The relative expression was calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001).

Plasmid construction

For transient gene expression in N. benthamiana, all the cloned genes and the Cs02526 gene without SP (Cs02526ΔSP) were cloned into the PVX vector pGR106. BAX and GFP were used as positive and negative controls, respectively. To generate a construct for prokaryotic expression, the coding region of Cs02526 (SP removed) was ligated into the pET-32a vector. To analyze the subcellular localization of Cs02526 in plants, full-length Cs02526 and Cs02526ΔSP were cloned into the pCambia1300 vector. For RNAi experiments in C. shiraiana, the sense and antisense silence fragments of Cs02526 were ligated into a pSilent-1 vector. Constructs used for VIGS in N. benthamiana were generated using the TRV2 vector, and N. benthamiana cDNA was used for the gene fragment amplification. All constructs were sequenced to verify their accuracy (Sangon Biotech). The primers used in this study are listed in Supplementary Table S3.

Protein expression, purification, and western blotting

pET-32a-Cs02526 was transformed into E. coli BL21(DE3) cells. Protein expression was induced on the LB medium by adding 0.3 mm isopropyl-beta-D-thiogalactopyranoside for 24 h at 16°C. The Cs02526 protein was purified using affinity chromatography with Ni-NTA resin (Sangon Biotech) following the manufacturer's instructions. Protein concentration was determined using the BCA Protein Concentration Assay Kit (Beyotime Biotech, Shanghai, China).

Total protein was prepared by milling 200 mg agroinfiltrated leaves in 500 μL of lysis buffer consisting of 1 mm phenylmethanesulfonylfluoride and protease inhibitor cocktail (Beyotime Biotech). The total protein was centrifuged at 4 °C for 10 min at 15,000 × g, and the supernatant was transferred to new tubes. The samples were boiled for 10 min in 5× SDS loading buffer and then loaded on a gel for SDS polyacrylamide gel electrophoresis (SDS–PAGE). For western blotting, total proteins were separated using 12% SDS–PAGE and transferred to PVDF membrane using a transfer buffer (20 mm Tris, 150 mm glycine). The PVDF membrane was blocked in 5% (w/v) nonfat dry milk for 2 h at room temperature (RT) with shaking at 50 rpm. Anti-GFP was used as the primary antibody for incubation at 4 °C overnight, and goat anti-mouse IgG (H + L) conjugated to horseradish peroxidase was used as the secondary antibody (Proteintech Group, Wuhan, China). Protein bands were detected using the ECL substrate following the manufacturer's instructions (Beyotime Biotech).

Bioinformatics analysis

The secretory proteins were predicted using SignalP 5.0 (Almagro Armenteros et al. 2019a) in C. shiraiana genomic data. TMHMM 2.0 was used to analyze transmembrane domains, and those without transmembrane domains were used for further experiments (Krogh et al. 2001). Protein sequences were entered into Targetp 2.0 and PredGPI to filter out the mitochondrial and glycosylphosphatidylinositol anchored proteins (Pierleoni et al. 2008; Almagro Armenteros et al. 2019b). Finally, EffectorP 2.0 was used to predict potential effectors (Sperschneider et al. 2018). Cs02526 homologs in other species were searched using BlastP in NCBI. Multiple sequence alignment of Cs02526 and its homologs was performed using the Muscle algorithm. Phylogenetic dendrograms were constructed using the MEGA7 software with the neighbor-joining tree method. The phylogenetic tree was edited by iTOL (https://itol.embl.de/). The diagrams of protein structures in this study were illustrated by IBS 1.0 software. The sequences of homologs are listed in Supplementary Data S1.

Agroinfiltration in N. benthamiana

For transient gene expression in N. benthamiana, A. tumefaciens carrying various PVX vectors were cultured in LB medium at 28 °C in a shaking incubator at 220 rpm for 36 h. Cells were collected by centrifugation for 10 min at 5,000 × g and washed 3 times using MES buffer (10 mm MgCl2, 10 mm MES, 200 μm acetosyringone, pH 5.7). Resuspension was kept in darkness for 3 h at RT and infiltrated into the leaves of 4- to 6-wk-old N. benthamiana with appropriate concentrations (OD600 = 0.4 to 0.8).

VIGS assays in N. benthamiana were based on the transient expression assay in N. benthamiana with modifications. The A. tumefaciens cell suspension carrying TRV2 constructs were mixed with A. tumefaciens cell suspension carrying TRV1 at a ratio of 1:1 before infiltration. pTRV2: GFP was used as the negative control. For these experiments, 3 biological replicates were conducted, with at least 6 plants with 3 inoculated leaves in each assay. The efficiency of gene silencing was determined through RT-qPCR analysis.

For subcellular localization in plants, A. tumefaciens carrying constructs were agroinfiltrated into 4-wk-old N. benthamiana leaves. The empty pCambia1300 vector was used as a negative control. GFP signals were observed using an FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan). The fluorescence of GFP was captured using the following excitation/emission settings: the GFP fluorescence was detected using a photomultiplier tube (PMT) detector (gain 498) at a wavelength ranging from 505 to 550 nm after argon laser excitation at 488 nm (intensity: 16.4%). Plasmolysis was performed by treating leaves with 30% (w/v) sucrose for 10 min.

Electrolyte leakage assay

To measure the ion leakage of N. benthamiana leaves, 6 leaf discs (9 mm diameter) were collected 7 d after agroinfiltration and floated in 4 mL distilled water for 5 h with continuous shaking (100 rpm) at RT. The conductivity of the solution was measured using a conductivity meter (FiveEasyPlus; Mettler-Toledo, Shanghai, China) to produce “value A.” Then, the leaf discs were boiled in sealed tubes containing a bathing solution for 30 min. When the solution was cooled to RT, the conductivity was measured to yield “value B.” The percentage of electrolyte leakage was calculated as (value A/value B) × 100%.

ROS detection and callose deposition

Leaves were stained with 1 mg/mL 3,3′-diaminobenzidine solution for 8 h in the dark and then destained with ethanol before observation for ROS accumulation. To visualize callose deposition, agroinfiltrated leaves were stained with aniline blue (Merck, Shanghai, China) according to a previous report (Shangguan et al. 2018).

Generation of RNAi strains and pathogenicity assay

To generate Cs02526 RNAi strains, transformants were obtained using the polyethylene glycol-mediated protoplast transformation method as described previously (Rollins 2003). All transformants were screened for consecutive generations on PDA containing 100 μg/mL hygromycin (Sangon Biotech). Positive transformants were screened using genomic PCR and were further confirmed by RT-qPCR to determine the relative expression levels of the target gene. To investigate the role of Cs02526 in C. shiraiana pathogenicity, the WT and RNAi strains were cultivated on PDA for 2 d. The 9 mm mycelial plugs were inoculated on N. benthamiana and mulberry leaves. The inoculated leaves were stored in high-humidity boxes. The lesion size was noted and photographed at 36 to 48 hpi. The biomass of C. shiraiana and S. sclerotiorum in the N. benthamiana leaves was determined by RT-qPCR as described previously. All experiments were conducted in at least 3 replicates and performed 3 times.

SIGS

The dsRNA used in this study was synthesized in vitro using the T7 High-efficiency Transcription Kit (TransGen Biotech, Beijing, China) following the manufacturer's instructions. For confocal microscopy examination of fluorescent dsRNA uptake by fungal mycelium, mycelium plugs (5 mm in diameter) were placed on slides containing PDA. Then, 10 μL of 100 ng/μL fluorescent dsRNA was added to the fungi and incubated for 12 to 18 h before confocal imaging. Mycelium was treated with KCl buffer (control) or 75 U micrococcal nuclease enzyme (dissolved in KCl buffer) to degrade the dsRNA on the surface of the mycelium before observation at 37 °C for 30 min. The fluorescent signal was analyzed using a confocal microscope (SpinSR, Olympus). The fluorescence was captured using the following excitation/emission settings: fluorescence was detected at a wavelength ranging from 565 to 615 nm after argon laser excitation at 555 nm (intensity: 25%). For dsRNA applied to C. shiraiana, 20 μL dsRNA (40 ng/μL) were dropped onto the surface of each plant sample. Then, 9-mm-diameter mycelium plugs were placed in the dsRNA-treated area. dsRNA-GFP was used as a negative control.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: Cs02526 (WLS32030.1), SS1G_07613 (XP_001590989.1), Bcnpc2 (XP_001558322.1), FGSG_09586 (XP_011328125.1), MGG_01557 (XP_003714532.1), and VDAG_00559 (XP_009650231.1).

Supplementary Material

kiae286_Supplementary_Data

Acknowledgments

We sincerely thank Yan Li (Analytical & Testing Center, Southwest University) and Dr. Shaoyu Zhang (State Key Laboratory of Resource Insects, Southwest University) for the assistance in confocal observations.

Author contributions

S.Z. and A.Z. conceived and designed the study. S.L. and R.L. provided help with the pathogenicity assays. W.F. and X.C. prepared the plant materials. P.Z., X.G., and S.W. provided technical assistance. S.Z. and A.Z. wrote and revised the manuscript. All authors have read and approved the final manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Screening and identification of effector proteins in C. shiraiana.

Supplementary Figure S2. Subcellular localization of Cs02526 and Cs02526ΔSP in N. benthamiana.

Supplementary Figure S3. Cs02526 could induce cell death in various plant species.

Supplementary Figure S4. Construction of VIGS experiments and detection of 02526 protein in N. benthamiana.

Supplementary Figure S5. Three-dimensional diagrams of Cs02526 and homologs.

Supplementary Figure S6. Cs02526 affected hyphal radial growth in C. shiraiana, but was dispensable for sclerotia development.

Supplementary Figure S7. Growth of WT and RNAi strains under different stress responses.

Supplementary Figure S8. Cs02526 was highly expressed during plant inoculation.

Supplementary Figure S9. Examination of the dsRNA uptake in C. shiraiana.

Supplementary Table S1. Candidate effector proteins of C. shiraiana screened in this study.

Supplementary Table S2. Information on Cs02526 and its homologs from 5 fungi.

Supplementary Table S3. Primers used in this study.

Supplementary Data S1. Cs02526 and its homolog sequence in this study.

Funding

This work was supported by the Hainan Province Science and Technology Special Fund (grant number ZDYF2022SHFZ319), the Earmarked Fund for China Agriculture Research System (grant number CARS-18-ZJ0201), and the Chongqing Modern Agricultural Industry Technology System (grant number CQMAITS202311).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

BAK1 Gramene: AT4G33430

BAK1 Araport: AT4G33430

FLS2 Gramene: AT5G46330

FLS2 Araport: AT5G46330

protein CHEBI: CHEBI:36080

PTI Gramene: pattern-triggered immunity

PTI Araport: pattern-triggered immunity

ANX1 Gramene: AT3G04690

ANX1 Araport: AT3G04690

ANX2 Gramene: AT5G28680

ANX2 Araport: AT5G28680
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