Molecular plant pathology最新文献

As the largest subfamily of small GTPases, the Rab subfamily plays a pivotal role in regulating biotic and abiotic stresses in plants. However, the functions of Rabs in resistance to wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) remain unclear. Here, we identified a Rab subfamily gene, TaRABH1bL, from Xiaoyan 6 (XY6), a wheat cultivar known for non-race-specific and durable high-temperature all-stage (HTAS) resistance to stripe rust. The expression level of TaRABH1bL was exclusively up-regulated with Pst inoculation under the relatively high-temperature treatment, which indicated that TaRABH1bL might concurrently respond to both biotic and abiotic stress signals. The TaRABH1bL gene was primarily expressed in leaves. Barley stripe mosaic virus (BSMV)-induced TaRABH1bL gene silencing significantly reduced HTAS resistance to Pst, resulting in increased sporulation. Transient expression of TaRABH1bL in Nicotiana benthamiana leaves and wheat protoplasts confirmed its subcellular localisation in both cytoplasm and nuclei. The GTP-binding state of TaRABH1bL (TaRABH1bL^Q69L) exclusively interacted with the transcription factor ethylene-responsive transcription factor 1-like (TaERF1L) in nuclei. TaERF1L directly bound to and suppressed the activity of the GCC-box motif, and this inhibitory effect was enhanced by the exclusive interaction between TaRABH1bL^Q69L and TaERF1L. Silencing TaERF1L significantly reduced HTAS resistance. These results suggested that under dual signals of Pst infection and relatively high temperature treatment, TaRABH1bL transferred into its GTP-binding state and interacted with TaERF1L. Additionally, TaRABH1bL^Q69L enhanced the suppression of TaERF1L on its downstream susceptible or temperature-sensitive genes containing the GCC-box motif, thereby activating HTAS resistance to Pst in XY6.

Phytophthora species are oomycetes that cause significant losses in agricultural production and damages to natural ecosystems. Phytophthora pathogens secrete numerous cytoplasmic effectors that target distinct cellular components to suppress host immunity and facilitate pathogen colonisation. The identification of their host targets is crucial for deciphering the mechanisms they employ to modulate host immunity. Here, we found that multiple Phytophthora Avr3a-like effectors interact with host plant cinnamyl alcohol dehydrogenase CAD5, as revealed by yeast two-hybrid and co-immunoprecipitation assays. Analysis of Arabidopsis thaliana T-DNA insertion mutants and overexpression lines, as well as analysis of RNA silencing Nicotiana benthamiana plants, showed that CAD5 positively regulates plant immunity to Phytophthora pathogens. Overexpression and silencing analyses showed that CAD5 plays a positive role in plant PAMP-triggered immunity (PTI) responses, including enhanced callose deposition, promoted cell death induced by INF1, and in plant effector-triggered immunity (ETI) responses mediated by R3a/PiAvr3a^KI recognition. CAD5 enzymatic activity was inhibited by Avr3a-like effectors, and mutagenesis analyses showed its crucial role in the positive regulation of plant immunity. In conclusion, our research showed that the Phytophthora Avr3a-like effectors target the conserved immune regulator CAD5 and suppress its enzymatic activity, which is required for both plant PTI and ETI responses.

Fungal effectors play crucial roles in plant infection. Despite low sequence identity, they were recently discovered to belong to families with similar three-dimensional structures. In this study, we elucidated the structures of Zt-NIP1 and Mycgr3-91409-2 effectors of the wheat fungal pathogen Zymoseptoria tritici using X-ray crystallography and NMR. These effectors displayed a structural homology with, respectively, KP4 and KP6α killer toxins from UmV dsRNA viruses of the maize fungal pathogen Ustilago maydis. Consequently, Zt-NIP1 and Mycgr3-91409-2 were renamed Zt-KP4-1 and Zt-KP6-1. Orthologues and paralogues of Zt-KP4-1 and Zt-KP6-1 were identified in Zymoseptoria, but not in other fungi, except ECP2 effectors related to Zt-KP4-1. Assessment of the biological activities of Zt-KP6-1 and Zt-KP4-1 revealed their ability to inhibit fungal growth, but they were unable to induce wheat leaf necrosis. A novel pipeline relying on cysteine-pattern constrained HMM searches and Foldseek analysis of AlphaFold2 predicted structures from Uniprot generated a comprehensive inventory of KP4 and KP6 proteins in fungi and plants. Their structure-based classification revealed four KP4 and three KP6 structural superfamilies and provided far-reaching hypotheses on their biological function and evolution. This framework highlights the power of structure determination and modelling for the classification of effectors and their functional investigation.

Potassium is an essential element for plant growth and development, and also plays a pivotal role in plant immunity against nematodes. However, it is not clear how plant nematodes manipulate host K⁺ signalling pathways to disrupt host immunity and promote their parasitism. Here, we demonstrate the rice K⁺ transporter OsHAK17 was targeted by the effector protein MgCOL from Meloidogyne graminicola nematodes. Genetic and phenotypic analyses revealed that knockout of OsHAK17 in rice resulted in decreased resistance to M. graminicola, while overexpression of OsHAK17 in rice enhanced resistance to M. graminicola. The yeast two-hybrid assay showed OsHAK17 interacted with MgCOL. In situ hybridisation assay showed MgCOL mRNA accumulating in the subventral oesophageal gland of J2 nematodes, while immunolocalisation analysis revealed MgCOL localises to the giant cells during M. graminicola parasitism. Host-induced gene silencing of MgCOL reduced the infection ability of M. graminicola, and over-expressing MgCOL enhanced rice susceptibility to M. graminicola. More importantly, MgCOL-overexpression rice showed decreased K⁺ accumulation in roots, which was consistent with it in nematode-infected rice. In conclusion, when M. graminicola nematodes infect rice and secrete MgCOL into rice, MgCOL interacts with OsHAK17, resulting in the change of K⁺ uptake and transportation in rice to enhance susceptibility.

Phelipanche aegyptiaca, a root holoparasitic weed, severely threatens agricultural productivity due to its detrimental effects. This species relies on a specialised organ, namely the haustorium, to extract nutrients from its host plants. The germination and haustorium formation of P. aegyptiaca are initiated by sensing host plant root exudates. Differences in exudate composition are crucial markers of host resistance. Host plant root exudates significantly influence the development and survival of P. aegyptiaca. To identify root exudates affecting the parasitic weed's growth, we analysed differential metabolites in resistant and susceptible Cucumis melo varieties. Among these, 6-hydroxynicotinic acid was identified as a key compound. Prehaustorium formation, which is induced by haustorium-inducing factors, such as indole-3-acetic acid (IAA), was suppressed in the presence of 6-hydroxynicotinic acid. This compound exerts an inhibitory effect by reducing the expression of genes related to the auxin signalling pathway of P. aegyptiaca, thus weakening parasitism. Our results support a model in which 6-hydroxynicotinic acid inhibits prehaustorium development in P. aegyptiaca by disrupting gene expression and endogenous metabolism.

The alterations in gene expression levels in response to the pathogens are pivotal in determining pathogenicity or susceptibility. However, the cell-type-specific interaction mechanism between the pinewood nematode (PWN) and its hosts remains largely unexplored and poorly understood. Here, we employed single-nucleus RNA sequencing (snRNA-seq) with PWN-infected Arabidopsis leaves to dissect the heterogeneous immune responses. We identified four major cell types, each exhibiting distinct immune responses during infection by PWNs. Subcluster analyses uncovered dynamic shifts in immune-active subpopulations within mesophyll and epidermal cells. Notably, AtWRKY70 positively regulated plant defence against PWNs by suppressing the promoter activity of AtPNP-A in a salicylic acid-dependent manner. This study not only provides novel mechanistic insights into plant gene regulation during PWN infection, but also offers feasible references for future investigations of host-PWN interactions, with particular relevance to the identification of pine tree resistance genes against this pathogen.

Southern corn leaf blight (SCLB), caused by Cochliobolus heterostrophus, is a destructive disease in maize-growing areas worldwide. Reactive nitrogen species derived from nitric oxide exhibit antimicrobial activities by interacting with microbial cellular components, leading to nitrosative stress in pathogens. However, the regulatory mechanisms underlying adaptation to nitrosative stress remain largely unexplored in C. heterostrophus. In this study, two components of the Rpd3 histone deacetylase complex, ChPho23 and ChSds3, were identified as being involved in the nitrosative stress response and virulence in C. heterostrophus. ChPho23 and ChSds3 are not only required for vegetative growth and conidiation but are also essential for responding to oxidative stress. ChPho23 and ChSds3 directly interact with ChHog1, and ChHog1 in turn interacts with ChCrz1 to up-regulate the transcription of genes involved in the nitrosative stress response, which enable C. heterostrophus to cope with nitrosative stress. Furthermore, mutants of ΔChhog1 and ΔChcrz1 exhibited significantly reduced virulence on detached maize leaves and increased sensitivity to nitrosative stress. Taken together, these findings indicated that ChPho23 and ChSds3 are crucial for fungal growth, conidiation, nitrosative stress response, and virulence in C. heterostrophus. This knowledge could be applied to the design of strategies that target ChPho23 and ChSds3 for controlling SCLB.

Downy mildew (DM) diseases are caused by destructive obligate pathogens with limited control options, posing a significant threat to global agriculture. RNA interference (RNAi) has emerged as a promising, environmentally sustainable strategy for disease management. We evaluated the efficacy of dsRNA-mediated RNAi in suppressing key biological functions in DM pathogens of Arabidopsis thaliana, pea and lettuce: Hyaloperonospora arabidopsidis (Hpa), Peronospora viciae f. sp. pisi (Pvp) and Bremia lactucae (Bl), respectively. Conserved genes, cellulose synthase 3 (CesA3) and beta-tubulin (BTUB), were targeted. Silencing these genes significantly impaired spore germination and infection across species and reduced gene expression correlated with suppressed sporulation, confirming silencing efficacy. We tested dsRNAs from chemical synthesis, in vitro transcription, and Escherichia coli expression. Uptake and silencing efficiency varied with dsRNA length and concentration. In Hpa, short dsRNAs (21-25 bp) produced a variable spore germination rate, with 25 bp dsRNA causing a 247.10% increase, whereas longer dsRNAs (≥ 30 bp) completely inhibited germination. Similarly, in Pvp, dsRNAs of 21-25 bp resulted in a 73.05%-77.46% germination rate, while 30-75 bp dsRNAs abolished germination. Confocal microscopy using Cy-5-labelled short-synthesised dsRNA (SS-dsRNA) confirmed uptake by spores. Sequence specificity influenced efficacy, highlighting the need for precise target design. Multiplexed RNAi impacted silencing synergistically, further reducing germination and sporulation in Hpa. Importantly, SS-dsRNA-mediated silencing was durable, with reduced gene expression sustained at 4, 7, 10 and 11 days post-inoculation. Taken together, our findings demonstrate the potential of dsRNA-mediated gene silencing as a precise, sustainable tool for managing DM pathogens in multiple crop species.

Panax vietnamensis, a medicinally valuable perennial herb, is highly susceptible to leaf blight under cultivation; however, the molecular mechanisms underlying this disease remain poorly understood. In this study, we identified Neofusicoccum ribis as the causal agent of P. vietnamensis leaf blight through pathogen isolation and fulfilment of Koch's postulates. Transcriptomic analysis revealed activation of phytohormone signalling (salicylic acid, jasmonic acid, and melatonin [MT]) and phenylpropanoid metabolism during infection. Among these, MT exhibited superior efficacy in inducing lignin biosynthesis compared to other hormones, with exogenous application of MT significantly enhancing lignin accumulation and improving disease resistance by 8 days post-inoculation. Further, we identified PvWRKY40 as a negative regulator of lignin synthesis, which directly binds to the W-box motif in the PvCOMT2 promoter to suppress its expression. MT counteracted this repression by downregulating PvWRKY40. Heterologous overexpression of PvCOMT2 in Nicotiana benthamiana increased lignin content and conferred enhanced resistance to Fusarium oxysporum. This study reveals a novel MT-PvWRKY40-PvCOMT2 regulatory axis governing lignin-mediated defence in P. vietnamensis, providing critical insights for combating leaf blight in cultivated ginseng.

Ceratocystis fimbriata is a destructive fungal pathogen that infects various economic crops. Nevertheless, the infection mechanism of this fungus is still unclear. Our previous studies have shown that the transcription factor CfSwi6 downstream of the cell wall integrity pathway is involved in regulating the pathogenicity of C. fimbriata. To further clarify the pathogenic mechanism of this pathway, upstream MAPKs (CfBck1-CfMkk1-CfSlt2) were characterised in this study. Deletion of CWI-MAPK genes resulted in an almost complete loss of pathogenicity of C. fimbriata. Importantly, CWI-MAPKs are associated with the formation of hyphopodia, which are infection structures required for C. fimbriata, and are reported for the first time in this work. Mutants lacking CWI-MAPK genes had defects in forming hyphopodia. The ability of mutants to penetrate cellophane membranes and host cells was reduced. CWI-MAPKs or CfSwi6 deletion affected CfSep4 assembly at penetration pegs, while CfSep4 was important for septin-ring and penetration peg formation. These results indicate that CWI-MAPKs regulate infection structure formation by modulating septin-ring organisation. RNA-seq analysis revealed that some downstream genes co-regulated by CfSlt2 and CfSwi6 are cellophane surface-induced genes. Knockout of PHH50197 and CfHSP30_1, two CfSlt2-CfSwi6-dependent genes, affected hyphopodium formation and pathogenicity. Additionally, other downstream genes, including PHH51274, CfHSP30_0, CfSTE11 and PHH55780, are not necessary for hyphopodium morphogenesis but are important for pathogenicity. Our study reveals a molecular mechanism by which CWI-MAPKs regulate pathogenicity through downstream genes mediated by CfSwi6 in C. fimbriata.