Molecular plant pathology最新文献

The tomato chlorosis virus (ToCV) is one of the most destructive plant viruses affecting tomato crops, leading to significant agricultural losses. As an obligate parasite, ToCV depends on the macromolecular machinery of host cells for replication. The ubiquitin 26S proteasome system maintains the intracellular protein homeostasis, which is essential for plant growth and development. Our study found that the CPm protein of ToCV interacted with SlPAD1, a component of the 26S proteasome, to enhance viral infection. This interaction disrupts the binding between SlPAD1 and SlPA4, thereby impairing the 26S proteasome function. In addition, SlPAD1 and SlPA4 positively regulate plant resistance to ToCV. Our findings reveal a mechanism by which ToCV proteins facilitate infection by interfering with 26S proteasome function.

Current challenges in controlling phytopathogenic bacteria lie in widespread chemical resistance, biosafety concerns, and the scarcity of novel biomacromolecule targets. While transposable elements have emerged as critical drivers of genetic variability and virulence in plant pathogens, their potential as druggable targets remains unexplored. Here, we report the first discovery of ISXoo15 transposase in Xanthomonas oryzae pv. oryzae (Xoo) as the bactericidal receptor for J9, a pyrimidine-substituted pleuromutilin derivative. In vitro assays demonstrate J9's superior anti-Xoo activity, with an EC₅₀ of 0.12 mg/L-significantly lower than commercial agents thiodiazole copper (86.39 mg/L) and zinc thiazole (26.15 mg/L). In vivo pot trials reveal enhanced curative and protective efficacy of J9 against rice bacterial leaf blight compared to these metal-based controls. A photoaffinity probe, P-J9, is synthesised and coupled with activity-based protein profiling to unequivocally identify ISXoo15 transposase (encoded by PXO_03433) as J9's specific target. Reverse transcription-quantitative PCR confirmed significant downregulation of PXO_03433 expression in J9-treated Xoo. Physiological and virulence-related functional analyses of a homologous recombination-mediated PXO_03433-knockout strain (ΔPXO_03433) showed markedly attenuated virulence and impaired pathogenicity. Conversely, PXO_03433-complemented strain CΔPXO_03433 possessed substantial restoration of pathogenicity-related traits. Proteomic profiling revealed significant downregulation of pathways associated with DNA repair, recombination and binding proteins in both J9-treated and mutant strains. ISXoo15 transposase may serve as a key regulator in enabling the homeostasis of the DNA metabolic network in the bacteria. This study provides pioneering evidence for targeting bacterial transposases as a novel antibacterial strategy, establishing a foundation for effective management of phytopathogenic bacteria.

Amino acid uptake is crucial for the pathogenicity of Puccinia striiformis f. sp. tritici (Pst), the causative agent of wheat stripe rust. In this study, we investigated the dynamics of cystine accumulation in wheat leaves during Pst infection and identified Pst cystine transporters involved in this process. Amino acid profiling revealed a marked increase in cystine content at early infection stages. Phylogenetic analysis and expression profiling identified four candidate Pst cystine transporter genes, among which PstAZ2B02G00053 (designated as PstCYN1) was functionally validated through yeast complementation assays. Subcellular localisation studies confirmed PstCYN1 as a plasma membrane transporter. Silencing of PstCYN1 via BSMV-VIGS and RNAi significantly reduced Pst virulence, as evidenced by decreased fungal biomass, reduced haustorial formation and fewer urediniospore pustules. Furthermore, apoplastic cystine accumulation and reactive oxygen species (ROS) levels were elevated in PstCYN1-silenced plants, indicating that PstCYN1 mediates not only cystine uptake but also redox regulation at the infection interface. These findings highlight the critical role of PstCYN1 in Pst nutrient acquisition and defence suppression, providing potential targets for enhancing wheat resistance against stripe rust.

RNA silencing is one of the major defence activities against viral pathogens in plants. Silencing signals are initiated by Dicer-like proteins (DCLs) to generate viral-derived small RNAs (sRNAs). Viral sRNAs are then loaded into Argonaute proteins to form an RNA-induced silencing complex to guide cleavage of target RNAs based on sequence homology. While the model regarding RNA silencing-mediated defence against viral pathogens is largely established based on extensive studies using the model plant Arabidopsis thaliana, there are diverse sets of silencing components in other plants, especially in domesticated crops. Here, we tracked the expansion of solanaceous-specific DCL2 genes during the course of evolution. We found that the DCL2a gene in tomato chromosome 6 is likely an evolutionarily new gene copy. We also found that DCL2b is more prone to be induced by viral pathogens in tomato plants, which is dependent on the combinations of cultivar and viral pathogen. Both DCL2a and DCL2b are critical to suppress the accumulation titre of a subviral agent, potato spindle tuber viroid (PSTVd). We noticed an unusually high accumulation of viral sRNAs shorter than 20 nt (16- to 19-nt in length) in viroid-infected tomato cv. Heinz 1706. Using synthetic small interfering RNAs, we demonstrated that shorter size sRNAs may also play a role in suppressing target RNAs, which can be interfered with by a viral suppressor of silencing, P19. Altogether, we provided further insights into the expansion of functional DCL2 family members in the Solanaceae family and their roles in combating viral and subviral agents.

Plants cope with the biotic stress caused by pathogen infection through complex resistance mechanisms. Here, we identified a secreted laccase Cglac8 from Colletotrichum gloeosporioides and confirmed its involvement in C. gloeosporioides infection of mango (Mangifera indica) plants, as well as its ability to activate the host's innate immune mechanism. Cglac8 interacted with MiLRR-RLP1 and Mi14-3-3-D1 as demonstrated by yeast two-hybrid, bimolecular fluorescence complementation and pull-down assays. The interacting proteins MiLRR-RLP1 and Mi14-3-3-D1 positively regulate mango resistance to C. gloeosporioides by promoting the reactive oxygen species burst and biosynthesis of phytohormones. When Cglac8, MiLRR-RLP1 and Mi14-3-3-D1 proteins were overexpressed together in mango, the resistance of mango to C. gloeosporioides was significantly enhanced. Our findings reveal a new defence mechanism of host plants against C. gloeosporioides, providing a theoretical basis for disease-resistant molecular breeding. The dual role of secretory laccase Cglac8 may reflect a balancing mechanism in host-pathogen co-evolution.

The necrotrophic fungal pathogen Rhizoctonia solani anastomosis group 3 (AG3-TB) is a major cause of global tobacco crop yield losses. Secreted proteins produced by filamentous fungi, as important virulence factors, play a core role in the interaction between plants and pathogens. In this study, we identified a secretory protein, RsDN3377, which localised to the intercellular space and induced cell death in Nicotiana benthamiana. Heterologous expression in Escherichia coli coupled with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis confirmed RsDN3377 possessed deacetylase activity. In addition, RsDN3377 was an essential pathogenicity factor for mycelial development by double-stranded RNA-mediated gene silencing. Through yeast two-hybrid and bimolecular fluorescence complementation assays, we demonstrated that RsDN3377 interacted with the calcium-binding protein NtCML19. In addition, transgenic Yunyan 87 overexpressing NtCML19 exhibited enhanced resistance to R. solani AG3-TB infection. Microscale thermophoresis analysis verified the calcium-binding activity of NtCML19. These lines of evidence indicate that the deacetylase RsDN3377 is secreted by R. solani AG3-TB, and this protein, critical for promoting fungal mycelial development and pathogenicity, was disrupted by its resistance-related interaction with NtCML19.

Lasiodiplodia theobromae can cause severe diseases, including leaf spot, leaf necrosis and stem canker in tea plants, leading to substantial losses in both tea leaf production and quality. However, the mechanisms underlying host resistance remain poorly understood. In this study, we identified CsWRKY57 as a nucleus-localised transcription factor whose expression dynamically responds to L. theobromae infection. Transient overexpression and antisense oligonucleotide (AsODN)-mediated silencing in tea leaves, along with stable overexpression in transgenic Nicotiana benthamiana leaves, demonstrated that CsWRKY57 enhanced resistance to L. theobromae. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that CsWRKY57 interacts with CsTIFY5A in the nucleus, further confirming that CsTIFY5A negatively regulates disease resistance through transient overexpression and AsODN-mediated silencing in tea leaves. DNA affinity purification sequencing, electrophoretic mobility shift assay and dual-luciferase assay indicated that CsWRKY57 binds to the AGTCAA motif in the CsLRR-RLK promoter, thereby activating its expression. Transient overexpression and AsODN-mediated silencing assays in tea leaves demonstrated that CsLRR-RLK positively regulates resistance to L. theobromae. Additionally, degradome sequencing, β-glucuronidase and dual-luciferase assays revealed that miR5368-p5 cleaves CsWRKY57 mRNA. Transient overexpression and AsODN assays in tea leaves, as well as stable overexpression of miR5368-p5 in transgenic N. benthamiana, indicated that miR5368-p5 negatively regulates resistance to L. theobromae. Our results suggest that the miR5368-p5-CsWRKY57-CsLRR-RLK module, which also includes CsTIFY5A interacting with CsWRKY57, plays a critical role in regulating the defence response of tea plants to L. theobromae. The results provide valuable insights into the mechanisms governing the response of tea plants to L. theobromae infection.

Pseudomonas syringae pv. tabaci (Pta) is an important plant pathogen, which causes wildfire disease in Nicotiana species. However, the genetic basis underlying strain-level differences in virulence remains largely unresolved. To address this, we performed a comparative genomic analysis between a highly virulent strain Pta6605 and a less virulent strain Pta7375. Despite high overall genome similarity, we identified key single-nucleotide polymorphisms, including premature stop-codon mutations in seven open reading frames in Pta7375. Notably, point mutations in two regulatory genes, such as fleQ, which encodes a transcription factor essential for flagellar biogenesis and biofilm formation, and gcbB, which encodes a GGDEF domain-containing diguanylate cyclase responsible for cyclic dimeric guanosine monophosphate (c-di-GMP) synthesis, were implicated in virulence disparity. Functional analyses using deletion and locus replacement mutants in the Pta6605 background revealed that the disruption of fleQ markedly reduced motility, flagellin production, c-di-GMP accumulation, biofilm formation and virulence level mirroring the Pta7375 phenotype. The gcbB replacement mutant showed reduced disease symptom development, although c-di-GMP levels remained comparable to the Pta6605 wild type. Locus replacement between strains confirmed that a point mutation in fleQ was the primary driver of reduced motility and flagellin expression in Pta7375. These findings indicate that the reduced virulence of Pta7375 is associated with impaired regulation of flagella-related genes and disruption of the FleQ-mediated c-di-GMP signalling, underscoring the value of comparative genomics in disentangling the complex regulatory networks that govern virulence in plant pathogens.

Plants typically encode multiple ubiquitin-activating enzymes (E1s or UBAs), but their functional equivalence or divergence remains unclear. Here, we demonstrate that the two tomato (Solanum lycopersicum) E1s, SlUBA1 and SlUBA2, differentially regulate development and immunity. Knockdown of SlUBA1 or SlUBA2 caused distinct growth and developmental defects in tomato, while silencing both genes resulted in severe abnormalities, rapid etiolation, and plant death within 5-7 weeks. Notably, silencing SlUBA2, but not SlUBA1, compromised plant immunity against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). SlUBA1 and SlUBA2 exhibited distinct charging efficiencies for E2s from groups IV (SlUBC32/33/34), V (SlUBC7/14/35/36), VI (SlUBC4/5/6/15) and XII (SlUBC22), with SlUBA2 showing significantly higher efficiency. Swapping the C-terminal ubiquitin-folding domains (UFDs) between SlUBA1 and SlUBA2 largely reversed their E2-charging efficiency for these groups. Furthermore, mutating a key residue (SlUBA2^Q1009) in the UFD or deleting a conserved 13-amino-acid sequence unique to group V E2s altered the E2-charging profiles of both E1s. These findings suggest dual ubiquitin-activating systems (DUAS) operate in tomato. Given the established role of group IV E2s in plant immunity against Pst, the SlUBA2-group IV E2 module likely plays a central role in modulating host defence. Similarly, the Arabidopsis E1s, AtUBA1 and AtUBA2, differentially charge homologues of tomato group IV E2s, suggesting a conserved mechanism by which plant E1s fulfil distinct physiological roles.