Necrotrophic pathogens cause serious threats to agricultural crops, and understanding the resistance genes and their genetic networks is key to breeding new plant cultivars with better resistance traits. Although Alternaria alternata causes black spot in important leafy brassica vegetables, and leads to significant loss of yield and food quality, little is known about plant-A. alternata interactions. In this study, we used a unique and large collection of single, double and triple mutant lines of defence metabolite regulators in Arabidopsis to explore how these transcription factors and their epistatic networks may influence A. alternata infections. This identified nine novel regulators and 20 pairs of epistatic interactions that modulate Arabidopsis plants' defence responses to A. alternata infection. We further showed that the glucosinolate 4-methoxy-indol-3-ylmethyl is the only glucosinolate consistently responsive to A. alternata infection in Col-0 ecotype. With the further exploration of the regulators and the genetic networks on modulating the accumulation of glucosinolates under A. alternata infection, an inverted triangle regulatory model was proposed for Arabidopsis plants' defence responses at a metabolic level and a phenotypic level.

Plant-pathogenic bacteria colonise their hosts using various strategies, exploiting both natural openings and wounds in leaves and roots. The vascular pathogen Xanthomonas campestris pv. campestris (Xcc) enters its host through hydathodes, organs at the leaf margin involved in guttation. Subsequently, Xcc breaches the hydathode-xylem barrier and progresses into the xylem vessels causing systemic disease. To elucidate the mechanisms that underpin the different stages of an Xcc infection, a need exists to image bacterial progression in planta in a non-invasive manner. Here, we describe a phenotyping setup and Python image analysis pipeline for capturing 16 independent Xcc infections in Arabidopsis thaliana plants in parallel over time. The setup combines an RGB camera for imaging disease symptoms and an ultrasensitive CCD camera for monitoring bacterial progression inside leaves using bioluminescence. The method reliably quantified bacterial growth in planta for two bacterial species, that is, vascular Xcc and the mesophyll pathogen Pseudomonas syringae pv. tomato (Pst). The camera resolution allowed Xcc imaging already in the hydathodes, yielding reproducible data for the first stages prior to the systemic infection. Data obtained through the image analysis pipeline was robust and validated findings from other bioluminescence imaging methods, while requiring fewer samples. Moreover, bioluminescence was reliably detected within 5 min, offering a significant time advantage over our previously reported method with light-sensitive films. Thus, this method is suitable to quantify the resistance level of a large number of Arabidopsis thaliana accessions and mutant lines to different bacterial strains in a non-invasive manner for phenotypic screenings.

The begomoviral V2 protein is known to be multifunctional, including its interaction with and inhibition of CYP1, a papain-like cysteine protease (PLCP). However, the effect of this interaction on viral pathogenicity remains unclear. Cotton leaf curl Multan virus (CLCuMuV), a typical monopartite begomovirus associated with a betasatellite, is one of the main pathogens responsible for cotton leaf curl disease. This study verifies the interaction between CLCuMuV V2 and NbCP15, a PLCP homologue in Nicotiana benthamiana. The results show that V2 can be cleaved by NbCP15 in vitro, with the N-terminal cleavage site located between the second and third amino acids. Using an Agrobacterium-mediated inoculation method, we investigated the influence of cleavage sites on viral pathogenicity. The findings indicate that mutation of the third amino acid in V2 (V2) reduced the pathogenicity of both heterologous PVX and CLCuMuV. Additionally, the NbCP15 gene mutation in N. benthamiana (nbcp15) also resulted in reduced CLCuMuV pathogenicity. These results suggest that CLCuMuV V2 may promote viral infection through its interaction with plant PLCPs.

Apple bitter rot is caused by various Colletotrichum spp. that threaten apple production globally resulting in millions of dollars in damage annually. The fungus causes a decline in fruit quality and yield, eventually rotting the fruit and rendering it inedible. The pathogen is difficult to keep out of orchards because of its broad host range and transmissibility by rain splash and insects. Once the disease manifests, pathogen identification is difficult due to evolving taxonomy and similar morphology between species. Current management strategies are threatened by an increase in fungicide resistance and regulations on many multisite fungicides, leading to a pressing need for new management options for control. This review aims to summarise the most current knowledge regarding the biology, virulence factors, ecology, omics and emerging management strategies for Colletotrichum species that cause apple bitter rot.

Tomato yellow leaf curl Guangdong virus (TYLCGdV), a monopartite begomovirus first identified in 2004, remains poorly characterised. In this study, we demonstrate that TYLCGdV associates with a betasatellite, TYLCGdB, and the βC1 protein encoded by TYLCGdB is essential for symptom development. We also explore the role of TYLCGdV C4 protein by generating a C4-deficient infectious clone (TYLCGdV), revealing a dynamic role for TYLCGdV C4. Specifically, viral accumulation in TYLCGdV/TYLCGdB-inoculated plants was significantly lower than that in TYLCGdV/TYLCGdB-inoculated plants at 7 and 14 days post-inoculation (dpi), but surpassed that of TYLCGdV/TYLCGdB-inoculated plants by 25 dpi. Furthermore, although C4 proteins in other begomoviruses typically exhibit one or more of the following properties: (i) suppression of post-transcriptional gene silencing (PTGS), (ii) suppression of transcriptional gene silencing (TGS), (iii) enhancement of pathogenicity in potato virus X (PVX) and (iv) symptom induction when transgenically expressed, TYLCGdV C4 did not exhibit any of these properties. However, the dynamic role of TYLCGdV C4 in viral infection appears to result from its effects on viral DNA methylation. At 7 dpi, the cytosine methylation level in the TYLCGdVmC4 genome was notably elevated compared to that of the wild-type virus. However, this trend reversed by 14 dpi, with the wild-type virus exhibiting a higher methylation level. By 25 dpi, the cytosine methylation levels of both TYLCGdVmC4 and TYLCGdV were comparable. These results indicate that TYLCGdV C4 modulates viral infection via an unconventional mechanism. This novel observation highlights the need for further investigation into the diverse roles of C4 proteins in begomoviruses.

Bacterial blight of cotton (BBC) caused by Xanthomonas citri pv. malvacearum (Xcm) is an important and destructive disease affecting cotton plants. Transcription activator-like effectors (TALEs) released by the pathogen regulate cotton resistance to the susceptibility. In this study, we sequenced the whole genome of Xcm Xss-V-18 and identified eight tal genes: seven on the plasmids and one on the chromosome. Deletion and complementation experiments of Xss-V-18 tal genes demonstrated that Tal1b is required for full virulence on cotton. Transcriptome profiling coupled with TALE-binding element prediction revealed that Tal1b targets GhSWEET14A04/D04 and GhSWEET14D02 simultaneously. Expression analysis confirmed the independent inducibility of GhSWEET14A04/D04 and GhSWEET14D02 by Tal1b, whereas GhSWEET14A04/D04 is additionally targeted by Tal1. Moreover, β-glucuronidase and Xa10-mediated hypersensitive response assays indicated that the effector-binding element (EBEs) are required for the direct and specific activation of the candidate targets by Tal1 and Ta1b. These insights enhance our understanding of the underlying mechanisms of bacterial blight in cotton and might lead to improved resistance through EBEs disruption or a TALE-trap strategy.

Cotton Verticillium wilt (VW) is often a destructive disease that results in significant fibre yield and quality losses in Gossypium hirsutum. Transferring the resistance trait of Gossypium barbadense to G. hirsutum is optional but challenging in traditional breeding due to limited molecular dissections of resistance genes. Here, we discovered a species-diversified structural variation (SV) in the promoter of receptor-like protein 6 (RLP6) that caused distinctly higher expression level of RLP6 in G. barbadense with the SV than G. hirsutum without the SV. Functional experiments showed that RLP6 is an important regulator in mediating VW resistance. Overexpressing RLP6 significantly enhanced resistance and root growth, whereas the opposite phenotype appeared in RLP6-silenced cotton. A series of experiments indicated that RLP6 regulated reactive oxygen species (ROS) and salicylic acid (SA) signalling, which induced diversified defence-related gene expression with pathogenesis-related (PR) proteins and cell wall proteins enrichments for resistance improvement. These findings could be valuable for the transfer of the G. barbadense SV locus to improve G. hirsutum VW resistance in future crop disease resistance breeding.

In the coevolutionary process between plant pathogens and hosts, pathogen effectors, primarily proteinaceous, engage in interactions with host proteins, such as plant transcription factors (TFs), during the infection process. This review delves into the intricate interplay between TFs and effectors, a key aspect in the prolonged and complex battle between plants and pathogens. Effectors strategically manipulate TFs using diverse tactics. These include modulating activity of TFs, influencing their incorporation into multimeric complexes, directly changing TF expression levels, promoting their degradation via the ubiquitin-proteasome system, and inducing their subcellular relocalization. The review systematically presents documented interactions, elucidating key mechanisms and their profound impact on host-pathogen dynamics. It emphasises the central role of TFs in plant defence and investigates the convergent evolution of effectors targeting TFs. By providing this overview, we offer valuable insights into this dynamic interaction landscape and suggest potential directions for future research.

Two phylogenetically unrelated viruses transmitted by different insect vectors, tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV), are major threats to tomato and other vegetable production. Although co-infections of TSWV and TYLCV on the same host plant have been reported on numerous occasions, there is still lack of research attempting to elucidate the mechanisms underlying the relationship between two viruses when they coexist in the same tomato or other plants. After assessing the effect of four TSWV-coded proteins on suppressing TYLCV in TSWV N transgenic Nicotiana benthamiana seedlings, the TSWV N protein proved to be effective in reducing TYLCV quantity and viral symptoms. Western blot analysis indicated that TSWV N was involved in down-regulating the expression level of the V1, C3, and C4 proteins of TYLCV, among which V1 was the most significantly suppressed one. Moreover, TSWV N was confirmed to reduce TYLCV V1 within both nucleus and cytoplasm, but a greater suppression was observed in cytoplasm. The co-immunoprecipitation and mass spectrometry identified 244 differential proteins from the TYLCV-infected TSWV N transgenic N. benthamiana seedling. These proteins pertaining to energy metabolism pathways were enriched, suggesting that TSWV N could inhibit TYLCV through competing for energy or regulating energy-related metabolism. The evidence presented here offers a novel perspective that will facilitate a comprehensive understanding of virus-virus and virus-host interactions, as well as a potential strategy for plant virus control through using TSWV N in the near future.

Zhu, L.-J., Zhu, Y., Zou, C., Su, L.-Y., Zhang, C.-T., Wang, C. et al. (2024) New persistent plant RNA virus carries mutations to weaken viral suppression of antiviral RNA interference. Molecular Plant Pathology, 25, e70020. Available from: https://doi.org/10.1111/mpp.70020 The following errors have been identified in the above published article: The co-first authors and co-corresponding authors are not indicated. The order of the funds appearing in the Funding information of the article are not consistent with the order in the Acknowledgements. The authors would like to correct these errors as follows: Li-Juan Zhu and Yu Zhu contributed equally to this work. Jian-Guo Wu and Yan-Hong Han are co-corresponding authors. Jiang-Guo Wu: wujianguo81@126.com; Yan-Hong Han: yan-hong@fafu.edu The correct order of funds is: National Natural Science Foundation of China, Grant/Award Number 32025031 and 31,900,153, National Key Research and Development Program of China, Grant/Award Number 2023YFF1000500 and Special Fund Project for Science and Technology Innovation of FAFU, Grant/Award Number KFB23013. We apologise for these errors.

Fungal plant pathogens cause major crop losses worldwide, with many featuring compartmentalised genomes that include both core and accessory regions, which are believed to drive adaptation. The highly host-specific fungus Colletotrichum lupini greatly impacts lupin (Lupinus spp.) cultivation. This pathogen is part of clade 1 of the C. acutatum species complex and comprises four genetically uniform, presumably clonal, lineages (I-IV). Despite this, variation in virulence and morphology has been observed within these lineages. To investigate the potential sources of genetic variability in this asexual fungus, we compared the genomes of 16 C. lupini strains and 17 related Colletotrichum species. Phylogenomics confirmed the presence of four distinct lineages, but further examination based on genome size, gene content, transposable elements (TEs), and deletions revealed that lineage II could be split into two groups, II-A and II-B. TE content varied between lineages and correlated strongly with genome size variation, supporting a role for TEs in genome expansion in this species. Pangenome analysis revealed a highly variable accessory genome, including a minichromosome present in lineages II, III, and IV, but absent in lineage I. Accessory genes and effectors appeared to cluster in proximity to TEs. Presence/absence variation of putative effectors was lineage-specific, suggesting that these genes play a crucial role in determining host range. Notably, no effectors were found on the TE-rich minichromosome. Our findings shed light on the potential mechanisms generating genetic diversity in this asexual fungal pathogen that could aid future disease management.

Many gram-negative pathogenic bacteria use type III effector proteins (T3Es) as essential virulence factors to suppress host immunity and to cause disease. However, in many cases the molecular function of T3Es remains unknown. The plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on tomato and pepper plants and is known to translocate around 36 T3Es into its host cell, which collectively suppress plant defence and promote infection. XopM is an Xcv core T3E with unknown function that has no similarity to any other known protein. We found that XopM interacts with vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) in an isoform-specific manner. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of membrane contact sites involved in both tethering and lipid transfer by binding directly to proteins containing an FFAT (two phenylalanines [FF] in an acidic tract [AT]) motif. Sequence analyses revealed that XopM displays two FFAT motifs that cooperatively mediated the interaction of XopM with VAP. When expressed in plants, XopM supported growth of a nonpathogenic bacterial strain and dampened the production of reactive oxygen species, indicating its ability to suppress plant immunity. Further analyses revealed that the interaction with VAP and the ability to suppress microbe-associated molecular pattern-triggered immunity (MTI) are structurally and functionally separable, although XopM requires localisation to the host membrane system for full MTI suppression activity. We discuss a working model in which XopM uses FFAT motifs to target the membrane to interfere with early MTI responses.

The organs of a plant species vary in cell structure, metabolism and defence responses. However, the mechanisms that enable a single pathogen to colonise different plant organs remain unclear. Here we compared the transcriptome of the oomycete pathogen Phytophthora sojae during infection of roots versus leaves of soybeans. We found differences in the transcript levels of hundreds of pathogenicity-related genes, particularly genes encoding carbohydrate-active enzymes, secreted (effector) proteins, oxidoreductase-related proteins and transporters. To identify the key regulator for root-specific infection, we knocked out root-specific transcription factors (TFs) and found the mutants of PsBZPc29, which encodes a member of an oomycete-specific class of basic leucine zipper (bZIP) TFs, displayed reduced virulence on soybean roots but not on leaves. More than 60% of the root-specific genes showed reduced expression in the mutants during root infection. The results suggest that transcriptional regulation underlies the organ-specific infection by P. sojae, and that a bZIP TF plays a key role in root-specific transcriptional regulation.

Microbial pathogens and other parasites can modify the development of their hosts, either as a target or a side effect of their virulence activities. The plant-pathogenic bacterium Ralstonia solanacearum, causal agent of the devastating bacterial wilt disease, is a soilborne microbe that invades host plants through their roots and later proliferates in xylem vessels. In this work, we studied the early stages of R. solanacearum infection in the model plant Arabidopsis thaliana, using an in vitro infection system. In addition to the previously reported inhibition of primary root length and increase in root hair formation at the root tip, we observed an earlier xylem differentiation during R. solanacearum infection that occurs in a HrpG-dependent manner, suggesting that the pathogen actively promotes the development of the vascular system upon invasion of the root. Moreover, we found that the phytohormone auxin, of which the accumulation is promoted by the bacterial infection, is required for the R. solanacearum-triggered induction of root hair formation but not earlier xylem differentiation. Altogether, our results shed light on the capacity of R. solanacearum to induce alterations of root developmental pathways and on the role of auxin in this process.

Viroids are single-stranded circular noncoding RNAs that mainly infect crops. Upon infection, nuclear-replicating viroids engage host DNA-dependent RNA polymerase II for RNA-templated transcription, which is facilitated by a host protein TFIIIA-7ZF. The sense-strand and minus-strand RNA intermediates are differentially localised to the nucleolus and nucleoplasm regions, respectively. The factors and function underlying the differential localisation of viroid RNAs have not been fully elucidated. The sense-strand RNA intermediates are cleaved into linear monomers by a yet-to-be-identified RNase III-type enzyme and ligated to form circular RNA progeny by DNA ligase I (LIG1). The subcellular compartment for the ligation reaction has not been characterised. Here, we show that LIG1 and potato spindle tuber viroid (PSTVd) colocalise near the nucleolar region in Nicotiana benthamiana protoplasts. The colocalised region is also the highly condensed region of sense-strand PSTVd RNA, indicating that PSTVd RNA and LIG1 form a biomolecular condensate for RNA processing. This finding expands the function of biomolecular condensates to the infection of subviral pathogens. In addition, this knowledge of viroid biogenesis will contribute to exploring thousands of viroid-like RNAs that have been recently identified.

Autophagy, one of the most widespread and highly conserved protein degradation systems in eukaryotic cells, plays an important role in plant growth, development and stress response. Beclin 1 is a core component of the phosphatidylinositol 3-kinase (PI3K) autophagy complex and positively regulates plant immunity against viruses. The upregulation of Eureka lemon ClBeclin1 was observed in response to citrus yellow vein clearing virus (CYVCV) infection. However, the function of ClBeclin1 and the underlying mechanism during CYVCV colonisation remain unclear. Here, the resistance evaluation of the overexpression and silencing of ClBeclin1 in Eureka lemon hairy roots revealed it as a positive regulator of citrus immunity against CYVCV. Transcriptomic profiling and metabolic analyses along with genetic evidence implied that the overexpression of ClBeclin1 positively triggered reactive oxygen species (ROS)- and jasmonic acid (JA)-mediated immunity in citrus. The accumulation of ROS and JA contents was attributed to the autophagic degradation of the ROS scavenger ClAPX1 via ClBeclin1 overexpression. Exogenous application of either HO or JA significantly reduced CYVCV colonisation and vein-clearing symptoms on the host. Collectively, our findings indicate that ClBeclin1 activation contributes to citrus immunity against CYVCV through triggering ROS- and JA-mediated defence responses, and the accumulation of ROS and JA resulted from the autophagic degradation of ClAPX1 by ClBeclin1.

Streptomyces scabies is a well-researched plant pathogen belonging to the genus Streptomyces. Its virulence is linked to the production of the secondary metabolite thaxtomin A, which is tightly regulated at the transcriptional level. The leucine-responsive regulatory protein (Lrp) family is conserved in prokaryotes and is involved in various crucial biological processes. However, the regulatory interaction between Lrp protein and pathogenic Streptomyces species remains poorly understood. This study aims to explore the role of SCAB_Lrp2 in regulating thaxtomin biosynthesis and pathogenicity, and to analyse the shared and unique features of Lrp homologues in S. scabies. We observed that SCAB_Lrp2 (SCAB_75421) showed significant homology with SCAB_Lrp, a recognised activator of thaxtomin A production in S. scabies. Our results revealed a regulatory interaction between SCAB_Lrp2 and SCAB_Lrp in terms of their targets, although SCAB_Lrp2 does not respond to the amino acid-effectors of SCAB_Lrp. In contrast to SCAB_Lrp, deletion of SCAB_Lrp2 resulted in a notable increase in thaxtomin A production with the emergence of a hypervirulent phenotype in S. scabies. Further analysis revealed that SCAB_Lrp2 represses the transcription of the thaxtomin biosynthetic gene cluster by directly regulating the cluster-situated regulator (CSR) gene txtR. Moreover, the precursor of thaxtomin, tryptophan, acts as an effector of SCAB_Lrp2, strengthening the repressive effect on thaxtomin biosynthesis through txtR. These findings provide new insights into the functional conservation and diversity of Lrp homologues involved in the biosynthesis of thaxtomin phytotoxins in pathogenic Streptomyces species.

Wei, Y.X., Liu, G.Y., Chang, Y.L., He, C.Z., Shi, H.T. Heat Shock Transcription Factor 3 Regulates Plant Immune Response Through Modulation of Salicylic Acid Accumulation and Signalling in Cassava. Molecular Plant Pathology, 2018; 19: 2209-2220. In the above article, there were unintentional errors in Figure 3 and Figure 6, specifically in the images of 0 dpi at Figure 3B and 6 dpi (pTRV-MeEDS1, pTRV-MePR4) at Figure 6C. These errors have been corrected in the below images: Corrected Figure 3 Corrected Figure 6 We apologise for these errors.

Cytokinin signalling plays both positive and negative roles in plant resistance to pathogens. It is not clear whether the role of cytokinin changes at the different stages of pathogen infection. Arabidopsis thaliana sequentially exhibits distinct root morphological symptoms during Ralstonia solanacearum infection, which offers a good system to investigate function of cytokinin in the whole pathogen infection process. Using this system, we found increase of cytokinin signalling by Lonely Guy 2 (LOG2) overexpression or depletion of type-A Arabidopsis Response Regulators (ARRs), negative regulators of cytokinin signalling pathway, promoted cell death, wilting symptom and bacterial growth, but attenuated primary root growth inhibition and lateral root formation. The decrease of cytokinin signalling by mutation on Isopentenyl Transferases (IPTs) inhibited root hair formation, cell death, wilting symptom and bacterial colonisation. Application of different concentration of exogenesis 6-benzylaminopurine (6-BA) showed first promoted, then decreased root hair formation. Moreover, application of 6-BA accelerated cell death but suppressed lateral root formation and primary root growth inhibition. The diverse roles of cytokinin in these different root disease phenotypes suggested function of cytokinin during plant responses to R. solanacearum is cell type-specific, which provides new insights on roles of cytokinin signalling in regulation on plant-pathogen interactions.

The compound appressoria of Sclerotinia sclerotiorum can produce cell wall-degrading enzymes, effectors and toxins, which promote penetration and the death of host cells. Subsequently, invasive hyphae (IH) branch rapidly as necrotrophic growth and disease symptoms are observed. S. sclerotiorum can respond to complex stresses and regulate its metabolism to adapt to the external environment. Here we demonstrated that type 2C Ser/Thr phosphatase (PP2C) SsPtc3 responds to nutritional, osmotic, cell wall and oxidative stresses. Loss of function ΔSsptc3 mutants displayed defects in mycelial growth, sclerotia formation and reduced virulence. Phosphoproteomic analyses revealed that SsPtc3 is involved in autophagy and MAPK signalling pathways. We obtained evidence that SsPtc3 negatively modulates the phosphorylation of SsSmk1. SsSmk1 is essential for mycelial growth, compound appressorium formation and pathogenicity, SsPtc3 modulated phosphorylation homeostasis of SsSmk1 to maintain hyphal growth. SsPtc3 interacted with SsAtg1 to influence autophagic flux under starvation. Taken together, these results reveal that SsPtc3 responds to various stresses that modulate autophagy and phosphorylation of SsSmk1-MAPK, which facilitates the growth and virulence of S. sclerotiorum.

In Arabidopsis thaliana, the transcription factors WRKY7, WRKY11 and WRKY17 act as negative defence regulators against Pseudomonas syringae pv. tomato (Pst) DC3000. However, their coordinated regulation of gene expression has yet to be fully explored. In this study, we conducted a transcriptomic analysis on the triple mutant wrky7/11/17 in response to Pst DC3000 at 0, 3 and 24 h post-inoculation (hpi). Our results suggest that at early infection stages (0 and 3 hpi), WRKY7, WRKY11 and WRKY17 significantly repress a group of genes involved in signal perception and transduction, including receptor-like kinases. Furthermore, at later stages of interaction (24 hpi), these transcription factors induce genes related to the biosynthesis and signalling of the jasmonic acid (JA) pathway. Further infection experiments with Pst DC3000 in plants treated with methyl jasmonate (a JA analogue) and infections with Botrytis cinerea, a pathogen against which JA-mediated responses are crucial for effective defence, support this proposal. Moreover, we analysed the role of WRKY7, WRKY11 and WRKY17 in alternative splicing regulation. A comparison between differentially expressed (DEG) and spliced (DAS) genes revealed that over 80% of DAS events do not occur in conjunction with overall changes in gene expression. Alternative splicing events were found in genes with functions in splicing and the JA pathway, such as ALY4, PRP40A, JAZ3 and JAZ10. These results suggest that WRKY7, WRKY11 and WRKY17 can also participate in this layer of gene expression regulation to modulate immunity negatively.