Leaf development into the final size is spatiotemporally regulated by cell proliferation and expansion. The jujube witches' broom (JWB) phytoplasma effectors SJP1/2 induce small leaves with decreased cell numbers in jujube plants by promoting the accumulation of the CIN-TCP transcription factor ZjTCP2. However, the molecular mechanism underlying SJP1/2-mediated inhibition of leaf cell proliferation remains largely unknown. Here, we report that SJP1/2 repressed cell proliferation by manipulating ZjTCP2 to control the CYC/TB1-TCP transcription factor ZjTCP1-mediated pathway. Compared with the mature leaves of the control lines, overexpression of ZjTCP1 in Arabidopsis resulted in large leaves with increased cell numbers, which was in contrast to the findings in ZjTCP2-overexpressing transgenic jujube plants. Yeast one-hybrid, luciferase and reverse transcription-quantitative PCR assays revealed that ZjTCP2 directly targeted the ZjTCP1 promoter and downregulated its expression. Furthermore, SJP1 induced the degradation of ZjTCP1 protein. ZjTCP1 was highly expressed during early leaf growth and was positively correlated with cell proliferation-related genes, including ANGUSTIFOLIA3 (ZjAN3), AINTEGUMENTA (ZjANT) and B-type CYCLINS1;1 (ZjCYCB1;1). Further investigations confirmed that ZjTCP1 directly bound to and upregulated the expression of ZjAN3, ZjANT and ZjCYCB1;1, whereas these genes were significantly downregulated in both SJP1/2 and ZjTCP2 transgenic jujube plants and JWB phytoplasma-infected jujube trees compared with those in the controls. These results provide a new insight into the molecular mechanism of SJP1/2-induced small-leaf formation through the manipulation of the ZjTCP2-ZjTCP1 cascade in woody plants.

Begomoviruses transmitted by whiteflies cause severe crop losses worldwide. Individual strains or isolates have a narrower host range, but collectively begomoviruses infect a wide range of plants. Begomovirus genomes undergo frequent recombination and mutations that confer a selective advantage in interactions with specific host factors facilitating host range adaptation, resulting in the rapid emergence of new strains with adapted host range. In this study, we examined the processes by which the begomoviruses can acquire and lose hosts by exchanging fragments of the viral genomes between a variant of tomato leaf curl New Delhi virus only infecting cucumber (ToLCNDV-C), tomato leaf curl Karnataka virus only infecting tomato (ToLCKV-T), and a ToLCNDV strain infecting both tomato and cucumber (ToLCNDV-T&C). We mapped the region responsible for tomato host loss to a 63 nucleotide (nt) region in the C-terminal of the transcriptional activator/replication enhancer protein (TrAP/REn) regions of ToLCNDV. We tested known host proteins reported to interact with this region using the yeast two-hybrid approach and found divergence in interactions with host proteins PCNA and AGO1. Finally, we found that the TrAP/REn region of DNA-A in conjunction with DNA-B can confer ToLCKV-T the ability to weakly infect its non-host, cucumber, and ToLCNDV-C to infect its non-host, tomato. Our studies reveal that multiple complex intra-virus interactions between viral proteins and virus-host interactions govern infectivity, virus accumulation and symptom severity.

Sugarcane mosaic virus (SCMV) causes substantial yield losses worldwide, yet the molecular basis underlying resistance and susceptibility in sugarcane remains incompletely understood. Here, we performed time-resolved transcriptome profiling of two contrasting sugarcane genotypes, the SCMV-susceptible cultivar Badila and its resistant somatic mutant FG1, across five infection stages. Absolute quantification revealed rapid viral RNA replication in Badila, whereas FG1 showed early suppression followed by SCMV clearance. Comparative transcriptomic analyses showed that FG1 mounted a rapid and sustained defence-associated transcriptional response, whereas Badila displayed delayed, predominantly repressive gene expression changes. Weighted gene co-expression network analysis identified gene modules strongly correlated with viral RNA levels and highlighted the small heat shock protein gene ScHSP17.5 as a central hub associated with susceptibility. Protein-protein interaction assays demonstrated that ScHSP17.5 and ScHSP17.9A specifically interact with the SCMV movement protein P3N-PIPO, but not with P3 or the coat protein. Functional assays in Nicotiana benthamiana further showed that overexpression of either ScHSP enhanced SCMV RNA replication, with co-expression producing a synergistic effect. Together, these results support a model in which SCMV exploits host small heat shock proteins via P3N-PIPO to promote viral accumulation, whereas early redox- and signalling-associated responses restrict infection in resistant sugarcane. This study provides mechanistic insight into SCMV-host interactions and identifies candidate targets for resistance breeding.

Leptosphaeria maculans is a phytopathogenic fungus responsible for stem canker on Brassica napus. Its infectious cycle goes through an early phase of leaf infection and a late phase of colonisation and infection of the stem. The disease is mainly controlled by plant genetic resistances targeting a limited set of early fungal effector genes overexpressed during leaf infection and located in dynamic repeat-rich genomic regions. Thus, these resistances can be rapidly overcome by the pathogen. To find new sources of resistance, we focused on late effector genes, expressed during stem infection and located in gene-rich regions. A previous study revealed a quantitative resistance in the stem, partly relying on a gene-for-gene interaction with a late effector gene. In this study, we deciphered whether all late effector genes shared the same genomic and evolutionary characteristics and if they could be more stable than early effector genes, rendering the resistance they trigger more durable. In addition, as previous studies highlighted new criteria for selecting late effectors and suggested B. napus semi-winter genotypes as an interesting genetic pool for uncovering resistance sources, we selected six new late effector gene candidates and screened an enlarged panel of semi-winter genotypes. We revealed that early and late effector genes diverged for most of their genomic characteristics, supporting the hypothesis of late effector genes being more conserved. Moreover, we revealed new resistance sources to late effector genes, almost all belonging to the semi-winter genetic pool, validating their importance to uncover new resistance sources.

ABSTRACTThe chestnut blight fungus Cryphonectria parasitica and its hypovirus constitute a valuable model for investigating fungal pathogenesis and cross‐kingdom virus–host interplay. To investigate how hypovirus regulates protein function at the phosphorylation level in C. parasitica, we performed a comparative phosphoproteomic analysis in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Comparative profiling between the wild‐type (EP155) and hypovirus‐infected (EP155/CHV1‐EP713) strains revealed 700 differentially phosphorylated sites (174 upregulated, 526 downregulated). Among these, the serine 896 and 897 sites on the endoplasmic reticulum (ER) stress‐sensing protein CpIre1 drew our particular attention, as hypovirus‐induced phosphorylation targets. Western blot analysis showed that virus‐encoded p29, p40, and p48 proteins could promote CpIre1 phosphorylation. Site‐specific mutagenesis revealed that Ser‐896 and Ser‐897 are essential for CpIre1 phosphorylation, which regulates fungal phenotypic traits, virulence, and stress tolerance in C. parasitica. Reverse‐transcription‐quantitative PCR analysis of the ER stress marker genes CpHac1 and CpBip1 confirmed that CpIre1 and its phosphorylation are essential for a functional ER stress response. Notably, hypovirus replication was significantly impaired in phospho‐deficient CpIre1 mutants, showing about 40% reduction in viral RNA accumulation, whereas phospho‐mimic mutants maintained wild‐type levels of viral RNA. This indicates that efficient hypovirus accumulation requires functional phosphorylation of CpIre1. Our findings demonstrate that hypovirus‐induced phosphorylation of CpIre1 modulates fungal ER homeostasis, pathogenicity, and viral RNA accumulation, thereby revealing a mechanism through which the virus reprogrammes its host via targeted post‐translational modification.

The cell wall serves as a critical barrier in plant defence against pathogen infection, whereas various Fusarium fungi secrete cell wall-degrading enzymes (CWDEs) to facilitate hyphal infection. In this study, luvangetin, a coumarin compound isolated and identified from the root of Zanthoxylum avicennae, was found to affect the cell wall degradation capacity and pathogenicity of Fusarium verticillioides on maize. Enzymatic activity assays of secreted enzymes from F. verticillioides demonstrated that luvangetin significantly inhibited the activity of the fungal crude enzyme extract, with the highest inhibition (13.5%) observed on cellulase activity. It also impaired the enzymatic hydrolysis to straw, wheat bran and bagasse. Integrated transcriptomic, proteomic and invitro activity analyses collectively revealed that luvangetin binds to three critical sites (Y193, D571 and E575) of the glycoside hydrolase 3 family (GH3) β-glucosidase in Fusarium species. Gene knockout and overexpression mutants were generated to further demonstrate that FvBgls3 plays a critical role in the pathogenicity of F. verticillioides and that it is an important target of luvangetin. Luvangetin directly binds to the catalytic active centre of FvBgls3, thereby suppressing the activity of CWDEs in F. verticillioides and ultimately reducing its pathogenicity. This study is the first to report that a coumarin small molecule directly binds to and inhibitthe activity of GH3 family enzymes, revealing the molecular mechanism by which luvangetin directly inhibits cell wall degradation capacity, providing novel targets and strategies for future control of F. verticillioides.

Pathogens deploy effector proteins to manipulate host physiology and promote infection. YopJ family effectors are highly conserved across bacterial genera that cause crop diseases. Nucleotide-binding leucine-rich repeat receptors (NLRs) play a central role in direct or indirect recognition of effectors and trigger immune responses, including hypersensitive cell death (HR). Two NLRs, Nicotiana benthamiana homologues of Pseudomonas tomato race 1 (NbPtr1) and HOPZ-ACTIVATED RESISTANCE 1 (NbZAR1), were recently identified as independently recognising two YopJ family effectors, HopZ5 and AvrBsT/XopJ2. NbZAR1 also detects XopJ4 via the receptor-like cytoplasmic kinase XOPJ4 IMMUNITY 2 (JIM2). Here, we conducted Agrobacterium-mediated transient expression assays with 20 YopJ family effectors from five phytopathogenic bacterial genera and identified 12 YopJ family effectors that are recognised either by NbZAR1 or independently by NbZAR1 and NbPtr1. Furthermore, we show that YopJ family effector-induced HR is differentially suppressed by the deacetylase SUPPRESSOR OF AVRBST-ELICITED RESISTANCE 1, suggesting more than one mechanism for YopJ family effector recognition. This work provides the genetic basis of the recognition of YopJ family effectors in N. benthamiana and lays a foundation for the mechanistic study of NbZAR1/JIM2 and NbPtr1 mode of activation.

Fhb1 is the most widely used locus for Fusarium head blight (FHB) resistance in wheat, yet the mechanistic basis of its candidate gene, TaHRC, remains elusive. Here, we demonstrate that the protein from the resistant allele TaHRC-R localises to both the nucleus and cytoplasm, whereas the susceptible protein TaHRC-S is confined to the nucleus. Remarkably, only TaHRC-R triggered a reactive oxygen species (ROS) burst in planta, dependent on its extranuclear localisation. The N-terminal 21 amino acids that distinguish TaHRC-R from TaHRC-S were essential for its nuclear export and ROS induction. Within the nucleus, TaHRC-R formed heterodimers with TaHRC-S via its N-terminal 21 amino acids and central region, disrupting the formation of large, sparse TaHRC-S condensates and converting them into numerous smaller assemblies. These results reveal a dual, spatially coordinated mechanism whereby TaHRC-R promotes ROS production outside the nucleus while modulating nuclear condensate dynamics to counteract the susceptible allele. This compartmentalised functionality provides a molecular framework for Fhb1-mediated resistance and illustrates a novel paradigm of subcellular specialisation in plant immunity.

ABSTRACTChilli veinal mottle virus (ChiVMV) is the most important virus known to threaten chilli pepper (Capsicum annuum) growth and yield in Asia and Africa. Here, we identified the class III peroxidase gene CaPOD49 as a key regulator of chilli pepper immunity against ChiVMV. The CaPOD49 expression was strongly induced in response to ChiVMV infection at 48 h post‐inoculation in both resistant and susceptible pepper cultivars. Silencing CaPOD49 via virus‐induced gene silencing (VIGS) significantly increased disease severity, reduced plant survival rates and increased the disease index of ChiVMV infection. This susceptibility was associated with the elevated accumulation of reactive oxygen species (ROS), while the treatment with the ROS scavenger diphenyleneiodonium (DPI) recovered pepper resistance. Additionally, the defence‐related genes CaPR2, CaPR5 and CaPR10 were significantly suppressed in CaPOD49‐silenced plants, as well as increasing its lipid peroxidation level. Heterologous expression of CaPOD49 in yeast enhanced its oxidative stress tolerance, consistent with the protective effects demonstrated by its peroxidase activity in vitro assays. Our findings demonstrate that CaPOD49 positively regulates chilli pepper immunity by fine‐tuning ROS levels and activating defence responses, offering potential genetic targets for disease‐resistant chilli pepper breeding.

Safflower (Carthamus tinctorius), a versatile economic crop of the Asteraceae family used for both medicinal and oil purposes, is widely cultivated worldwide. During its growth period, particularly during the harvesting phase, safflower is susceptible to fungal infections, leading to reduced product quality and yield. Currently, there is a lack of research on candidate genes associated with safflower disease resistance, which hinders the breeding ofdisease-resistant varieties of safflower. The disease severity index of 499 safflower germplasm lines during the flowering period was analysed through genome-wide association studies (GWAS), and a highly correlated TLP (thaumatin-like proteins) family disease resistance candidate gene, CtTLP13, was identified. Subsequently, transcriptomic and proteomic analyses were conducted to characterise the molecular features of safflower under fungal infection, and it was confirmed that CtTLP13 can respond to the biotic stress when safflower is infected by Botrytis cinerea. CtTLP13 overexpression in Arabidopsis and safflower enhanced safflower disease resistance; invitro experiments confirmed its inhibition of B. cinerea growth and spore germination. We isolated safflower extracellular vesicles (EVs), verified CtTLP13 localisation within them and showed that CtTLP13-carrying safflower EVs are taken up by B. cinerea and inhibit the fungal growth. Overall, this study identified a valuable disease resistance gene (CtTLP13) in safflower. We also investigated the role of safflower EVs in disease resistance, and the results showed that safflower EVs exhibit anti-B. cinerea activity.