Comparative genome analysis of plant ascomycete fungal pathogens with different lifestyles reveals distinctive virulence strategies

Background Pathogens have evolved diverse lifestyles and adopted pivotal new roles in both natural ecosystems and human environments. However, the molecular mechanisms underlying their adaptation to new lifestyles are obscure. Comparative genomics was adopted to determine distinct strategies of plant ascomycete fungal pathogens with different lifestyles and to elucidate their distinctive virulence strategies. Results We found that plant ascomycete biotrophs exhibited lower gene gain and loss events and loss of CAZyme-encoding genes involved in plant cell wall degradation and biosynthesis gene clusters for the production of secondary metabolites in the genome. Comparison with the candidate effectome detected distinctive variations between plant biotrophic pathogens and other groups (including human, necrotrophic and hemibiotrophic pathogens). The results revealed the biotroph-specific and lifestyle-conserved candidate effector families. These data have been configured in web-based genome browser applications for public display (http://lab.malab.cn/soft/PFPG). This resource allows researchers to profile the genome, proteome, secretome and effectome of plant fungal pathogens. Conclusions Our findings demonstrated different genome evolution strategies of plant fungal pathogens with different lifestyles and explored their lifestyle-conserved and specific candidate effectors. It will provide a new basis for discovering the novel effectors and their pathogenic mechanisms.

Disclosure of salicylic acid and jasmonic acid-responsive genes provides a molecular tool for deciphering stress responses in soybean

Hormones orchestrate the physiology of organisms. Measuring the activity of defense hormone-responsive genes can help understanding immune signaling and facilitate breeding for plant health. However, different from model species like Arabidopsis, genes that respond to defense hormones salicylic acid (SA) and jasmonic acid (JA) have not been disclosed in the soybean crop. We performed global transcriptome analyses to fill this knowledge gap. Upon exogenous application, endogenous levels of SA and JA increased in leaves. SA predominantly activated genes linked to systemic acquired resistance and defense signaling whereas JA mainly activated wound response-associated genes. In general, SA-responsive genes were activated earlier than those responding to JA. Consistent with the paradigm of biotrophic pathogens predominantly activating SA responses, free SA and here identified most robust SA marker genes GmNIMIN1, GmNIMIN1.2 and GmWRK40 were induced upon inoculation with Phakopsora pachyrhizi, whereas JA marker genes did not respond to infection with the biotrophic fungus. Spodoptera exigua larvae caused a strong accumulation of JA-Ile and JA-specific mRNA transcripts of GmBPI1, GmKTI1 and GmAAT whereas neither free SA nor SA-marker gene transcripts accumulated upon insect feeding. Our study provides molecular tools for monitoring the dynamic accumulation of SA and JA, e.g. in a given stress condition.

Comparative secretomics identifies conserved WAxR motif-containing effectors in rust fungi that suppress cell death in plants

Identification of novel effectors with conserved features has always remained a challenge in plant-pathogen interaction studies. The introduction of the genomics era in plant-pathogen studies has led to the identification of significant candidate effectors with novel motifs such as RxLR and dEER motifs. However, in the case of fungal pathogens, limited conserved motifs associated with effectors have been discovered yet. In the present study, we have performed comparative secretome analysis for major plant pathogens of diverse nutrition mechanisms with the aim of dissecting the features underlying their corresponding secretome and conserved motifs. We showed that rust fungi possess the lowest Cell wall degrading enzymes (CWDEs) consortium lower than other biotrophic pathogens. We also showed rust fungi possess the highest secretory superoxide dismutase (SOD) than other studied plant pathogens. Further, we prioritized the candidate secretory effectors proteins (CSEPs) of all the studied pathogens by combining various effector mining parameters to highlight the candidates with potential effector features. A novel WAxR motif in conjugation with the Y/F/WxC (FGC) motif was identified in the effectors of various P. striiformis races present globally. The WAxR/WAxR like motifs ( WxxR, WAxx, xAxR) containing effectors were also found in the secretome of other rust fungi. Further, the functional validation of two candidate effectors with WAxR motif from P. striiformis Yr9 showed that these effectors localize to the nucleus as well as cytoplasm, and are able to suppress BAX induced cell death in Nicotiana benthamiana. The mutation analysis of individual residues of the WAxR motif (W, A, R ) however did not affect the cell death suppression nor subcellular localization of these effectors. Overall, the current study reports the presence of novel motifs in large numbers of effectors of rust fungi with cell death suppression features.

Long-term epigenetic memory of jasmonic acid-dependent immunity against herbivory

Stress exposure can have long-lasting impacts on ecologically relevant life-history traits in plants. Here, we have investigated the long-term impacts of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis thaliana. Three weeks after transient JA signalling activity in seedlings, 5-week-old plants retained induced resistance (IR) against herbivory but showed enhanced susceptibility to necrotrophic and biotrophic pathogens. Transcriptome analysis revealed that JA seedling treatment causes prolonged priming and/or up-regulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR against herbivory was dependent on MYC2/3/4 transcription factors and DNA (de)methylation pathways. Although DNA methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled defence genes, plants from JA-treated seedlings showed enrichment of differentially methylated regions at ATREP2 transposable elements (TEs). Our study points to a trans-acting mechanism whereby hypomethylated TEs mediate long-lasting epigenetic memory of JA-dependent immunity.

Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticola NLP 1–8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

Conserved Opposite Functions in Plant Resistance to Biotrophic and Necrotrophic Pathogens of the Immune Regulator SRFR1

Plant immunity is mediated in large part by specific interactions between a host resistance protein and a pathogen effector protein, named effector-triggered immunity (ETI). ETI needs to be tightly controlled both positively and negatively to enable normal plant growth because constitutively activated defense responses are detrimental to the host. In previous work, we reported that mutations in SUPPRESSOR OF rps4-RLD1 (SRFR1), identified in a suppressor screen, reactivated EDS1-dependent ETI to Pseudomonas syringae pv. tomato (Pto) DC3000. Besides, mutations in SRFR1 boosted defense responses to the generalist chewing insect Spodoptera exigua and the sugar beet cyst nematode Heterodera schachtii. Here, we show that mutations in SRFR1 enhance susceptibility to the fungal necrotrophs Fusarium oxysporum f. sp. lycopersici (FOL) and Botrytis cinerea in Arabidopsis. To translate knowledge obtained in AtSRFR1 research to crops, we generated SlSRFR1 alleles in tomato using a CRISPR/Cas9 system. Interestingly, slsrfr1 mutants increased expression of SA-pathway defense genes and enhanced resistance to Pto DC3000. In contrast, slsrfr1 mutants elevated susceptibility to FOL. Together, these data suggest that SRFR1 is functionally conserved in both Arabidopsis and tomato and functions antagonistically as a negative regulator to (hemi-) biotrophic pathogens and a positive regulator to necrotrophic pathogens.

Disclosure of Salicylic Acid and Jasmonic Acid-responsive Genes Provides a Molecular Tool for Deciphering Stress Responses in Soybean

Hormones orchestrate the physiology of organisms. Measuring the activity of defense hormone-responsive genes can help understanding immune signaling and facilitate breeding for plant health. However, different from model species like Arabidopsis, genes that respond to defense hormones salicylic acid (SA) and jasmonic acid (JA) have not been disclosed in the soybean crop. We performed global transcriptome analyses to fill this knowledge gap. Upon exogenous application, endogenous levels of SA and JA increased in leaves. SA predominantly activated genes linked to systemic acquired resistance and defense signaling whereas JA mainly activated wound response-associated genes. In general, SA-responsive genes were activated earlier than those responding to JA. Consistent with the paradigm of biotrophic pathogens predominantly activating SA responses, free SA and here identified most robust SA marker genes GmNIMIN1, GmNIMIN1.2 and GmWRK40 were induced upon inoculation with Phakopsora pachyrhizi, whereas JA marker genes did not respond to infection with the biotrophic fungus. Spodoptera exigua larvae caused a strong accumulation of JA-Ile and JA-specific mRNA transcripts of GmBPI1, GmKTI1 and GmAAT whereas neither free SA nor SA-marker gene transcripts accumulated upon insect feeding. Our study provides molecular tools for monitoring the dynamic accumulation of SA and JA, e.g. in a given stress condition.

Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid’s (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Tissue-specific mRNA profiling of the Brassica napus-Sclerotinia sclerotiorum interaction uncovers novel regulators of plant immunity

White mold in Brassica napus (canola) is caused by the fungal pathogen Sclerotinia sclerotiorum and is responsible for significant losses in crop yield across the globe. With advances in high-throughput transcriptomics, our understanding of the B. napus defense response to S. sclerotiorum is becoming clearer; however, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll and vascular leaf tissue layers in response to S. sclerotiorum. This strategy increases the number of genes detected compared to whole-leaf assessments and provides unprecedented information on tissue-specific gene expression networks in response to pathogen attack. Our findings provide novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. Using bioinformatics tools, we identified several defense genes that might coordinate plant immunity responses shared across different tissue layers within the leaf. These genes were functionally characterized by challenging T-DNA insertion lines of Arabidopsis with necrotrophic, hemi-biotrophic, and biotrophic pathogens, ultimately converging on the PR5-like RECEPTOR KINASE (PRK5). Together, these data provide insight on the complexity of the B. napus defense response directly at the site of infection.