Molecular Plant Pathology

Plasmodiophora brassicae causes a significant global threat to cruciferous vegetables and crops. However, the current comprehensions of its pathogenic ways is still unclear. This study identified a P. brassicae effector, called PbEGF1, which strongly induces cell death in N. benthamiana. Notably, PbEGF1 was significantly up-regulated in seedlings inoculated with highly virulent P. brassicae, indicating a pivotal role for PbEGF1 in pathogenicity. Furthermore, overexpression of PbEGF1 in hosts enhanced susceptibility to P. brassicae, and promoted elongation of root hairs, thus creating favorable conditions for root hair infection. Silencing of PbEGF1 reduced the pathogenicity of P. brassicae. This finding confirms the significance of primary infection in host recognition and interaction with P. brassicae. To further elucidate the virulence function of PbEGF1, we identified BnNHL13 (nonrace-specific disease resistance 1/harpin-induced 1-like 13) as its target protein. Silencing BnNHL13 enhanced host susceptibility to P. brassicae, and promoted root hairs elongation, indicating that down-regulation of BnNHL13 was more conducive to establishing P. brassicae infection. Subsequent investigation revealed that PbEGF1 has the ability to induce degradation of the BnNHL13 protein, thereby disrupting the host defense response and facilitating P. brassicae infection. Our findings provide novel insights into genetic strategies for enhancing plant resistance against clubroot disease.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious crops diseases. To facilitate infection, B. cinerea secretes a large number of effectors that induce plant cell death. In screening secretome data of B. cinerea during infection stage, we identified a phytotoxic protein (BcPTP1) that can also induce immune resistance in plants. BcPTP1 is a small (90 aa), cysteine rich protein without any known domains. Transiently expression of BcPTP1 in leaves caused chlorosis that intensifies with time and eventually lead to cell death. Point mutations in eight of the 10 cysteine residues of BcPTP1 abolished the toxic effect, however residual toxic activity remained after heating the peptide, suggesting contribution of unknown epitopes to protein phytotoxic effect. The transcript level of the bcptp1 gene was low during the first 36 h after inoculation and increased sharply upon transition to the late infection stage, suggesting a role of BcPTP1 in lesion spreading. While statistically insignificant, deletion of the bcptp1 gene led to slightly smaller lesions on bean leaves. Further analyses indicated that BcPTP1 is internalized into plant cells after secreting into the apoplast and its phytotoxic effect is negatively regulated by the receptor-like kinases BAK1 and SOBIR1. Collectively, our findings show that BcPTP1 is a virulence factor that toxifies the host cells and facilitates lesion spreading during the late infection stage.

Sclerotinia sclerotiorum causes white mold or stem rot in a broad range of economically important plants, bringing significant yield losses worldwide. Host-induced gene silencing (HIGS) has been showing promising effects in controlling many fungal pathogens, including S. sclerotiorum. However, molecular genetic understanding of signaling pathways involved in its development and pathogenicity is needed to provide effective host-induced gene silencing (HIGS) targets for disease control. Here, by employing a forward genetic screen, we characterized an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade in S. sclerotiorum, consisting of SsSte50-SsSte11-SsSte7-Smk1, controlling mycelial growth, sclerotia development, compound appressoria formation, virulence, and hyphal fusion. Moreover, disruption of the putative downstream transcription factor SsSte12 led to normal sclerotia but aberrant appressoria formation and host penetration defects, suggestive of diverged regulation downstream of the MAPK cascade. Most importantly, targeting of SsSte50 using host-expressed HIGS double stranded RNA resulted in largely reduced virulence of S. sclerotiorum on Nicotiana benthamiana leaves. Therefore, this MAPK signaling cascade is generally needed for its growth, development, and pathogenesis, and is an ideal HIGS target for mitigating economic damages caused by S. sclerotiorum infection.

During host-pathogen interactions, fungal pathogens secrete effector proteins into host cells to manipulate the host immune system and facilitate infection. Although many effector genes are highly expressed during infection stages, there is limited information on the mechanisms regulating their in planta expression. Here, we characterize the promoter of MoHTR1, a nuclear effector gene of the rice blast fungal pathogen, to elucidate its in planta-specific expression. Using promoter deletion and mutation analyses, we identified a core cis-element (TATTTCGT) within the MoHTR1 promoter, designated the in planta active (IPA) element, which is crucial for in planta-specific expression. The IPA element is responsible for the expression of not only MoHTR1, but also other effector genes including a known effector Slp1. Furthermore, the IPA element enables the in planta expression of MobZIP14, a gene specifically expressed during vegetative growth. The IPA element plays a critical role in fungal virulence by enabling MoHTR1 expression and regulating host immune responses. Bioinformatic and DNA-protein interaction analyses revealed that RGS1, a transcription factor containing a winged-helix binding domain, acts as a transcriptional regulator of MoHTR1 by directly binding to the IPA element. Our findings provide new insights into the regulatory mechanisms driving the in planta-specific expression of fungal effector genes.

The re-emergence of Gram-negative bacterium Xylella fastidiosa in Europe in 2013 impelled the scientific community to discover novel strategies for crop protection. The wide host range of Xylella indicates the existence of yet not characterized pathogenic mechanisms to overcome plant defenses. The recent uprising accuracy of a variety of bioinformatics tools, with the ability to predict the function of putative microbial protein represent a useful approach for understanding which of these proteins are associated with pathogens virulence. In this study we collected a number of putative effectors from two X. fastidiosa strains: Temecula1 and CoDiRo and the subspecies (ssp.) Sandyi Ann-1. We designed an in-planta Agrobacterium based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in various model plants. Furthermore, we organized the resulted proteins according to their sequential and structural similarities via the I-TASSER online tool. We identified that various X. fastidiosa proteins were able to differentially elicit cell death-like phenotypes in Nicotiana tabacum, N. sylvestris and N. benthamiana. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. Collectively, we identified structurally similar proteins that were able to differentially elicit cell death-like phenotypes in different cultivars of the same species. Our findings provide the bases for further studies on the mechanisms that underlie host-defense activation by X. fastidiosa putative effectors, as well as, pathogens adaptation in susceptible hosts.

O_LIColletotrichum gloeosporioides is the dominant causal agent of rubber tree anthracnose and leads to serious loss of natural rubber production. Fungi secrete numerous effectors to modulate host defense systems. Understanding the molecular mechanisms by which fungal effectors regulate plant defense is of great importance for the development of novel strategies for disease control. C_LIO_LIHere, we identified an NLP effector gene, CgNLP1, which contributed to virulence of C. gloeosporioides to rubber tree. Transient expression of CgNLP1 in the leaves of Nicotiana benthamiana induced ethylene production in plants. Ectopic expression of CgNLP1 in Arabidopsis significantly enhanced the resistance to Botrytis cinerea and A. brassicicola. C_LIO_LICgNLP1 was shown to target a R2R3 type transcription factor HbMYB8-like in rubber tree, which localized on nucleus and induced necrosis in N. benthamiana. CgNLP1 disrupted nuclear accumulation of HbMYB8-like and suppressed necrosis induced by HbMYB8-like mediated SA signal pathway. C_LIO_LIThis work suggested a strategy whereby C. gloeosporioides exploited CgNLP1 effector to suppress host defense to facilitate infection by disrupting the subcellular compartment of a host defense regulator HbMYB8-like. C_LI

Blufensin1 (Bln1) has been identified as a negative regulator of basal defense mechanisms that is unique to the cereal grain crops barley, wheat, and rice. However, the molecular mechanisms through which Blufensin1 regulates the wheat immune response are poorly understood. In this study, we found that TaBln1 is significantly induced by Puccinia striiformis f. sp. tritici (Pst) virulent race CYR31 infection. Knockdown the expression of TaBln1 by virus-induced gene silencing reduced Pst growth and development, and enhanced the host defense response. In addition, TaBln1 was found to physically interact with TaCaM3 on the plasma membrane. Silencing TaCaM3 with virus-induced gene silencing increased fungal infection areas and sporulation and reduced wheat resistance to the Pst CYR23 and CYR31. Moreover, we found that the TaCaM3 transcription level could be induced by treatment with chitin but not flg22. Silencing TaCaM3 decreased the Ca2+ influx induced by chitin, but silencing TaBln1 increased the Ca2+ influx in vivo using a non-invasive micro-test technique. Taken together, we identified the wheat negative regulator TaBln1, which interacts with TaCaM3 to impair Ca2+ influx and inhibits plant defenses. One-sentence summaryTaBln1 negatively regulate wheat resistance to stripe rust possibly due to the interaction with TaCaM3 on the plasma membrane, which impairs the calcium influx modulated by TaCaM3.

O_LILeaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which comprises five paralogous genes (Cf-9A-9E) that encode receptor-like proteins. Two of these proteins contribute to resistance: Cf-9C recognizes the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that have overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. C_LIO_LIComparative genomics, in planta transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a worldwide strain collection. C_LIO_LIA strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. C_LIO_LIThis research showcases how F. fulva has evolved to sequentially break down the two functional resistance genes of the complex Cf-9 locus and highlights that this locus now has limited value for controlling leaf mould disease in worldwide commercial tomato production. C_LI

O_LILysM effectors are suppressors of chitin-triggered plant immunity in biotrophic and hemibiotrophic fungi. Their role in necrotrophic fungi is unclear as these last are known to activate plant defenses and induce cell death. C_LIO_LITo characterize the role of the BcLysM1 gene encoding a putative LysM effector in the necrotrophic fungus Botrytis cinerea, its expression was followed by transcriptional fusion and by RT-qPCR in planta. Two tagged-recombinant proteins were produced, and two independent deletion strains were constructed and characterized. C_LIO_LIBcLysM1 is induced in the early phase of infection, and more specifically in multicellular appressoria called infection cushions. The BcLysM1 protein binds the chitin in the fungus cell wall and protects hyphae against degradation by external chitinases. It is also able to sequester chitooligosaccharides and to prevent them from inducing ROS production in A. thaliana. Using mycelium as inoculum, deletion strains show a delay in infection initiation and a default in adhesion to bean leaf surfaces. C_LIO_LIThis study demonstrates for the first time a dual role for a LysM effector in mycelium adhesion on the plant and in host defenses suppression, both of them occurring during the asymptomatic phase of infection by a necrotrophic fungus. C_LI

Xanthomonas euvesicatoria (X. euvesicatoria) is the causal agent of bacterial spot disease that threatens pepper and tomato production around the globe. X. euvesicatoria gene Xe4428 encodes a type III effector (T3E) that shares 89.67% amino acid identity with Xanthomonas oryzae pv. oryzicola (Xoc) T3E AvrRxo1. Deletion of Xe4428 in the genome of X. euvesicatoria (strain Xcv85-10) compromised its virulence to infect pepper and Nicotiana benthamiana plants. Transient co-expression of Xe4428 and Rxo1 on pepper and N. benthamiana plant leaves results in a robust hypersensitive reaction. Thus, Xe4428, renamed as XeAvrRxo1, is a bona fide orthologue of XocAvrRxo1 that possesses both virulence and avirulence functions. Expression of XeAvrRxo1 in E. coli and X. euvesicatoria is toxic to both bacterial cells. Another X. euvesicatoria gene Xe4429, encodes a putative chaperone of XeAvrRxo1, which can interact with XeAvrRxo1 to suppress its toxicity in X. euvesicatoria and E. coli bacterial cells. Xe4429 also binds to the promoter region of XeavrRxo1 and represses its transcription/translation in X. euvesicatoria bacterial cells. In addition, expression of Xe4429 can enhance the secretion and translocation of XeAvrRxo1 into plant cells. Therefore, Xe4429 functions as an antitoxin, a transcription repressor, and a type III chaperone that is capable of enhancing the secretion and translocation of XeAvrRxo1 during pathogenesis.

Understanding the interactions between fungal plant pathogens and host roots is crucial for developing effective disease management strategies. This study investigates the molecular mechanisms underpinning the chemotropic responses of the maize anthracnose fungus Colletotrichum graminicola to maize root exudates. We identify the 7-transmembrane G-protein coupled receptor (GPCR) CgSte3 as a key player in sensing both plant-derived class III peroxidases and diterpenoids. Activation of CgSte3 initiates signaling through the Cell Wall Integrity Mitogen-Activated Protein Kinase (CWI MAPK) pathway, facilitating the pathogens growth towards plant defense molecules. The NADPH oxidase CgNox2 is crucial for peroxidase sensing but not for diterpenoid detection. These findings reveal that CgSte3 and CWI MAPK pathways are central to C. graminicolas ability to hijack plant defense signals, highlighting potential targets for controlling maize anthracnose.

Colletotrichum sublineola (Cs) is a hemibiotrophic fungal pathogen that causes anthracnose in Sorghum bicolor, leading to significant yield losses. To enable infection, Cs secretes effectors - proteins, small RNAs, and metabolites - that damage the plant cell wall or enter the plant cell to suppress immune responses and manipulate host metabolism. Effectors can detoxify host antimicrobials, alter nutrient processing, and evade host immunity. Paradoxically, some effectors can also trigger pattern-triggered immunity (PTI), especially in biotrophic and necrotrophic fungi. More than half of fungal protein effectors lack conserved domains and functional network annotations. In this study, we identified prospective Cs effectors, separating those with non-conserved domains and classifying those with conserved domains by protein families. Comparative genomics is employed to predict effector functions and analyze their roles. Using their predicted locations and domains, we mapped the effectors into functional subsystems related to PTI. These include interactions in the apoplast, oxidative stress response, protein modification and degradation systems, and Cysteine-rich Fungus-specific Epidermal Growth Factor-like Module (CFEM) domain proteins involved in immune regulation. Our functional network analysis advances the understanding of Cs pathogenicity and offers insights into effector infection mechanisms.

Xanthomonas fragariae (Xaf) is the cause for strawberry crown dry cavity rot and strawberry leaf angular spots. Despite having a long evolutionary history with strawberries, the plant-pathogen connection is poorly understood. Pathogenicity for the majority of plant pathogens is mostly dependent on the type-III secretion system, which introduces virulence type III effectors (T3Es) into eukaryotic hosts cells. For most of these T3Es, the subcellular targets are yet unclear. Here, We used the yeast-two-hybrid (Y2H) technique to construct an interaction network of strawberry-Xaf T3Es. Multiple T3Es were discovered to converge onto the strawberry 1-aminocyclopropane-1-carboxylic acid oxidases (ACOs), which are the last rate-limited step in the production of ethylene. We then concentrated on the connection between XopL and FveACO9. Strawberry plants that overexpressed XopL accumulated higher levels of ethylene and exhibited more severe Xaf infection. XopL boosted ethylene production by stabilizing the accumulation of FveACO9 protein and enhancing ACO enzyme activity. Additionally, strawberries treated with ACC or overexpressing FveACO9 were particularly vulnerable to Xaf infection. On the other hand, pre-treatment with -aminoinoisobutyric acid (AIB), an ACO inhibitor, effectively reduced Xaf infection. Our research indicates that Xaf utilizes a distinct approach to regulate the ethylene production of host plants in order to promote infection.

Potato brown rot, caused by Ralstonia solanacearum, is one of the most destructive diseases of potatoes. The pathogen could hide in the tuber, leading to the rotting tubers. However, few mechanisms of pathogenesis in tubers caused by brown rot were reported. Here, we identified a highly virulent type III effector RipBH, which is not only required for the pathogenesis of potato brown rot but also displays strong cell toxicity in yeast and tobacco. We found RipBH is a novel structural cysteine protease with a large ankyrin repeat domain that contains 10 ankyrin repeats, we named it as an ankyrin cysteine protease. Biochemical analysis showed that all the ankyrin repeats are required for virulence, and the first five ankyrin repeats are indispensable for auto-cleavage site recognition. Further analysis showed that RipBH triggered autophagy-associated cell death. The ankyrin cysteine protease effector existed extensively in plant and animal pathogens suggesting the ankyrin cysteine protease effectors are functionally essential for pathogen pathogenesis. Our study enhances our understanding of this type of cysteine protease and illustrates the pathogenesis of cysteine protease in potato brown rot.

Sclerotinia sclerotiorum induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification, oxalic acid production, pathogen establishment and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to necrotrophic pathogens and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper is transported into the necrotic area from healthy area during infection. Collectively, our data suggest that S. sclerotiorum hijacks host copper to detoxify ROS, whereas the resistant hosts restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

O_LITo date, translational regulation of key genes controlling infection-related processes in fungal pathogens during their interactions with plants has not been studied. Here, we employed ribosome profiling (ribo-seq) to study translational responses and how such responses are coordinated with transcriptional changes in the fungal pathogen Fusarium graminearum (Fg), which causes Fusarium head blight (FHB), a destructive disease of cereal crops worldwide. C_LIO_LITranscription and translation were not always coordinated with approximately 22% of Fg genes showing a discordant relationship during wheat infection. Nitrite reductase, which we show here as an important component of fungal virulence, is only regulated at the translational level in Fg. In addition, more than 1000 new open reading frames (ORFs), many of which are short and highly conserved, were identified in the Fg genome. C_LIO_LILike in higher eukaryotes, translation is controlled by upstream ORFs (uORFs) in Fg during infection. Similarly, miRNAs control both transcription and translation in Fg during wheat infection. However, Fgdicer2-dependent miRNAs do not have a significant effect on transcriptional gene expression at the global outset. C_LIO_LIThe ribo-seq study undertaken here for the first time in any fungal pathogen discovered novel insights about the biology of an important plant pathogen. C_LI

Plants have a sophisticated and multilayered immune system. However, plant pathogens, helped by effector proteins, have found several strategies to evade plant immunity. For instance, the clubroot pathogen, Plasmodiophora brassicae, is able to turn the roots of the susceptible hosts into nutrient-sink galls surpassing patterns-triggered immunity (PTI) and effector-triggered immunity (ETI). Chitin, the main component of P. brassicae spores cell walls and a well-known pathogens-associated molecular pattern (PAMP), can elicit PTI but is also the target of plant chitinases and chitin deacetylases. The fact that P. brassicae does not trigger PTI during the infection of the susceptible hosts motivated a genome-wide search of genes coding for secreted chitin-related proteins. We found that P. brassicae genome encodes a large repertoire of candidate-secreted effectors containing the chitin-binding domain carbohydrate-binding module family 18 (CBM18), along with chitinases and chitin deacetylases domains. The role of such proteins in the pathogenicity of the clubroot pathogen is unknown. Here, we characterized the function of two effectors, PbChiB2 and PbChiB4, which are transcriptionally activated during the spores transition to uninucleate primary plasmodium and during the spore formation. Through co-precipitation, we found that recombinant PbChiB2 and PbChiB4 bind to the spores and to chitin oligomers in vitro. We also showed that both proteins suppress chitin-triggered activation of the immune MPK3 and MPK6 in the host Brassica napus. These findings suggest a dual role for the P. brassicae CBM18 proteins as effectors for protecting zoospores and resting spores formation and for suppressing chitin-triggered immunity during the infection.

Clubroot caused by the soil-borne protist pathogen Plasmodiophora brassicae is one of the most devastating diseases of Brassica oil and vegetable crops worldwide. Understanding the pathogen infection strategy is crucial for the development of disease control. However, the molecular mechanism by which this pathogen promotes infection remains largely unknown. Here, we identified a P. brassicae-secreted effector PbE3-2 that impedes plant immunity by ubiquitinating the immune regulator RD21A for degradation. Overexpression of PbE3-2 in Arabidopsis thaliana resulted in higher susceptibility to P. brassicae and decreases in chitin-triggered reactive oxygen species burst and expression of marker genes in salicylic acid signaling. PbE3-2 interacted with and ubiquitinated RD21A in vitro and in vivo. Mutant plants deficient in RD21A exhibited similar susceptibility and compromsied immune responses as in PbE3-2 overexpression plants. These results suggest that P. brassicae promotes clubroot disease through RD21A degradation mediated by the effector PbE3-2. As PbE3-2 is widely conserved across different P. brassicae pathotypes, the degradation of RD21A by PbE3-2 might be a prevalent infection strategy in this pathogen.

Rust fungi are pathogens that impact plants of environmental, agricultural, cultural, and economic importance. Their mechanisms of pathogenicity are not well-understood but are likely governed by effectors, secreted proteins that manipulate host cellular processes to facilitate infection and suppress immune responses. We sought to understand how three effector candidates (EFC1, EFC2, and EFC3) expressed in the first stages of Austropuccinia psidii (myrtle rust) infection influence pathogenicity. We experimentally tested gene function through application of double-stranded RNA (dsRNA) and characterised the genomic landscape of putative effectors expressed during infection to assess whether putative effectors are needed for infection, and whether they are under selection pressure. One of the three screened candidates, EFC1, met our criteria of an effector in that it was predicted to be secreted, and was needed to cause but not maintain infection. We identified that this effector belongs to a gene family of intragenomic variants in tandem repeats flanked by transposable elements. Single nucleotide polymorphisms among these variants have signatures of non-neutral selection. This effector has predicted structural homology to a glycosaminoglycan-binding domain and may have a role in pectin or chitin-binding. We hypothesise that intragenomic variability in this family of effector genes facilitates host-range versatility in the A. psidii-Myrtaceae pathosystem.