Molecular Plant-Microbe Interactions®

Many phytopathogens translocate virulence (effector) proteins into plant cells to circumvent host immune responses during infection. One such pathogen is Pseudomonas syringae pv. tomato DC3000, which secretes at least twenty-nine effectors into host cells, of which a subset elicits host defense responses in crop plant species. However, it is unknown whether P. syringae pv. tomato DC3000 activates immune responses in diverse maize inbreds. Here, we screened a diverse maize germplasm collection for effector-dependent recognition of this bacterial pathogen. As a control, we infiltrated Pseudomonas syringae DC3000(D36E), a derivative of P. syringae pv. tomato DC3000 that lacks all endogenous effectors. In our evaluations, we observed a variety of responses to P. syringae pv. tomato DC3000 in maize and scored the phenotypes as either no observable response (N) or as one of three responses: weak chlorosis (WC), chlorosis (C) with minimal cell death, and hypersensitive reaction (HR)-like cell death. Of the twenty-six maize inbreds screened, 13 were scored as N, 2 as WC, 2 as C, and 9 as HR-like cell death. Importantly, no maize line responded to P. syringae DC3000(D36E), demonstrating the responses observed are likely dependent upon recognition of one or more Pseudomonas effectors. Importantly, maize inbreds that recognize P. syringae pv. tomato DC3000 accumulated detectable hydrogen peroxide as well as an increase in transcript expression of a subset of maize defense genes. Collectively, our results will likely stimulate new research aimed at identifying the cognate maize disease resistance proteins that recognize the activities of one or more bacterial effectors.

Summary/Abstract Pseudomonas syringae is a diverse phytopathogenic species complex, and includes strains that can cause disease across a wide variety of plant species. Much previous research into the molecular basis of immunity and infection has focused on pathogen and plant responses in a handful of model strains and hosts, and with a tacit assumption that early steps in infection and host resistance are generalizable to the species complex and across plant hosts as a whole. Here, we provide a test of this assumption by measuring the dual pathogen and host transcriptomes of two distinct pathogenic lineages of P. syringae during compatible infection of a shared model host (Nicotiana benthamiana). Our results demonstrate that, with a handful of exceptions, host plants largely respond in a similar way to both pathogenic lineages and both bacterial pathogens possess highly similar transcriptional responses at 5 hours post inoculation. However, we also highlight that subsets of genes with differential expression patterns in both bacteria and host which likely represent strain-specific responses.

Phyllachora maydis is an ascomycete foliar fungal pathogen and the causal agent of tar spot in maize. Though P. maydis is considered one of the most economically important foliar pathogens of maize, our general knowledge of the trophic lifestyle and functional role of effector proteins from this fungal pathogen remains limited. Here, we utilized a genome-informed approach to predict the trophic lifestyle of P. maydis and functionally characterized a subset of candidate effectors from this fungal pathogen. Leveraging the most recent P. maydis genome annotation and the CATAStrophy pipeline, we show this fungal pathogen encodes a predicted Carbohydrate-active enzymes (CAZymes) repertoire consistent with that of biotrophs (monomertrophs). To investigate fungal pathogenicity, we selected eighteen candidate effector proteins that were previously shown to be expressed during primary disease development. We assessed whether these putative effectors share predicted structural similarity with other characterized fungal effectors and determined whether any suppress plant immune responses. Using AlphaFold2 and Foldseek, we showed one candidate effector, PM02_g1115, adopts a predicted protein structure similar to that of an effector from Verticillium dahlia. Furthermore, transient expression of candidate effector-fluorescent protein fusions in Nicotiana benthamiana revealed that most effector proteins localize to both the nucleus and the cytosol. Importantly, three candidate effectors consistently attenuated chitin-mediated reactive oxygen species production in N. benthamiana. Collectively, these results presented herein provide valuable insights into the predicted trophic lifestyle and putative functions of effectors from P. maydis and will likely stimulate continued research to elucidate the molecular mechanisms used by P. maydis to induce tar spot.

O_LITranscription Activator-Like Effector (TALE) Tal12a is widespread in strains of Xanthomonas campestris pv. campestris (Xcc) causing black rot on Brassica crops. We sought to determine whether and how Tal12a contributes to disease. C_LIO_LITranscriptomic analysis of cauliflower leaves infected with Xcc strains expressing Tal12a was combined with TALE-binding element prediction and heterologous expression assays in Nicotiana benthamiana in order to identify candidate Susceptibility (S) genes. Artificial TALEs (arTALEs) were used to validate the contribution of candidate target genes to susceptibility. C_LIO_LITal12a enhances bacterial virulence and growth in cauliflower. Transcriptomic analysis revealed 380 upregulated cauliflower genes, from which nine were selected as candidate targets. Expression of sugar transporter genes BoSWEET13 and BoSWEET14c was induced though likely indirectly. Five genes were confirmed as direct targets of Tal12a. Functional assays showed that BoIAA7c auxin-dependent transcriptional regulators and BoSWEET14c each independently contribute to disease, but not to bacterial proliferation. C_LIO_LIThis work identifies the first susceptibility genes in cauliflower. Tal12a enhances susceptibility in cauliflower thanks to a complex transcriptional reprogramming comprising both direct and indirect tal12a targets, some of which act as minor S genes. C_LI

Xanthomonas perforans is the predominant pathogen responsible for bacterial leaf spot of tomato and X. euvesicatoria of pepper in the southeast United States. Previous studies have indicated significant changes in the X. perforans population collected from Florida tomato fields over the span of two decades including a shift in race, diversification into three genetic groups, and host range expansion to pepper. Recombination originating from X. euvesicatoria was identified as the primary factor driving the diversification of X. perforans in Florida. The aim of this study was to genetically characterize X. perforans strains that were isolated from tomato and pepper plants grown in Alabama and compare them to the previously published genomes available from GenBank. Surprisingly, a maximum likelihood phylogeny coupled with a Bayesian analysis of population structure revealed the presence of two novel genetic groups in Alabama, which each harbored a different transcription activation-like effector (TALE). While one TALE, avrHah1, was associated with adaptation of X. perforans to pepper, the other was identified as a new class within the avrBs3 family, designated here as pthXp1. Examination of patterns of homologous recombination between X. perforans and other closely related Xanthomonas spp. indicated that the lineages identified here emerged in part through recent recombination events originating from xanthomonads associated with diverse hosts of isolation. Our results also suggest that the evolution of pathogenicity to pepper has likely emerged independently within X. perforans and in one lineage, was associated with the recombination-mediated remodeling of the Xps type II secretion and TonB transduction systems.\n\nImportanceThe emergence of novel pathogen lineages has important implications in the sustainability of genetic resistance as a disease management tool in agricultural ecosystems. In this study, we identified two novel lineages of X. perforans in Alabama. While one lineage was isolated from symptomatic pepper plants, confirming the host range expansion of X. perforans, the other lineage was isolated from tomato and acquired a novel transcription activation-like effector, pthXp1. Unlike AvrBs4, PthXp1overcomes Bs4-mediated resistance in tomato, indicating the evolution of this novel lineage towards fitness on this host. Our findings also show that different phylogenetic groups of the pathogen have experienced independent recombination events originating from multiple Xanthomonas species. This suggests a continuous gene flux between related xanthomonads associated with diverse plant hosts which results in the emergence of novel pathogen lineages and associated phenotypes, including host range expansion.

Zymoseptoria tritici is the most economically significant fungal pathogen of wheat in Europe. However, despite the importance of this pathogen, the molecular interactions between pathogen and host during infection are not well understood. Herein, we describe the use of two libraries of cloned Z. tritici effectors that were screened to identify effector candidates with putative pathogen associated molecular pattern (PAMP) triggered immunity (PTI)-suppressing activity. The effectors from each library were transiently expressed in Nicotiana benthamiana, and expressing leaves were treated with bacterial or fungal PAMPs to assess the effectors ability to suppress reactive oxygen species (ROS) production. From these screens, numerous effectors were identified with PTI-suppressing activity. In addition, some effectors were able to suppress cell death responses induced by other Z. tritici secreted proteins. We used structural prediction tools to predict the putative structures of all of the Z. tritici effectors, and used these predictions to examine whether there was enrichment of specific structural signatures among the PTI-suppressing effectors. From among the libraries, multiple members of the killer protein-like 4 (KP4) and killer protein-like 6 (KP6) effector families were identified as PTI-suppressors. This observation is intriguing, as these protein families were previously associated with antimicrobial activity rather than virulence or host manipulation. This data provides mechanistic insight into immune suppression by Z. tritici during infection, and suggests that similar to biotrophic pathogens, this fungus relies on a battery of secreted effectors to suppress host immunity during early phases of colonisation.

Alternaria blight or leaf spot caused by Alternaria brassicae has an enormous economic impact on the Brassica crops grown worldwide. Although the genome of A. brassicae has been sequenced, little is known about the genes that play a role during the infection of the host species. In this study, the transcriptome expression profile of A. brassicae during growth and infection was determined. Differential expression analysis revealed that 3921 genes were differentially expressed during infection. Weighted gene co-expression network analysis helped identify nine modules, which were highly correlated with growth and infection. Subsequent gene ontology (GO) enrichment analysis of the modules highlighted the involvement of biological processes such as toxin metabolism, ribosome biogenesis, polysaccharide catabolism, copper ion transport, and vesicular trafficking during infection. Additionally, 194 CAZymes and 64 potential effectors were significantly upregulated during infection. Furthermore, 17 secondary metabolite gene clusters were also differentially expressed during infection. The clusters responsible for the production of Destruxin B, Brassicicene C, and HC-toxin were significantly upregulated during infection. Collectively, these results provide an overview of the critical pathways underlying the pathogenesis of A. brassicae and highlight the distinct gene networks that are temporally regulated, resulting in a biphasic mode of infection. The study thus provides novel insights into the transcriptional plasticity of a necrotrophic pathogen during infection of its host. Additionally, the in planta expression evidence for many potential effectors provides a theoretical basis for further investigations into the effector biology of necrotrophic pathogens such as A. brassicae.

Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease and can cause up to 50% yield losses globally. Despite its economic impact, understanding of the molecular interactions between Z. tritici and its host remains limited, particularly the functions of many uncharacterized candidate effectors. To explore the roles of candidate effectors in modulating host immune responses, we selected seven Z. tritici genes with elevated expression during the early biotrophic phase and the transition to necrotrophy in a susceptible interaction. These candidates were transiently expressed in Nicotiana benthamiana, both with and without their predicted signal peptides. AlphaFold structural predictions revealed that two candidates share similarity with proteins of known function: a sterol-binding protein from Saccharomyces cerevisiae and a necrosis-inducing effector from Valsa mali. Effector activity did not always correlate with expression timing, and the presence of a signal peptide significantly influenced the activity of candidate effectors on host defense responses. Several effectors consistently attenuate the production of reactive oxygen species (ROS), while none suppress PBR1-mediated cell death, indicating they do not target this NLR or its downstream signaling. Two candidate effectors, Mycgr3107904 and Mycgr394290, induce cell death in N. benthamiana while also modulating the ROS burst, suggesting potential dual functions at different stages of infection. These findings provide new insights into how Z. tritici effectors modulate plant immunity during disease progression, either to evade host recognition or establish infection. Our results show that effector functions may extend beyond what is inferred from expression profiles alone.

Botrytis cinerea is a plant pathogen that causes significant agricultural losses worldwide. Although this necrotroph disrupts extensive plant hormonal networks, the role of salicylic acid (SA) in plant defense against B. cinerea remains controversial across plant species. To investigate its role from a pathogen perspective, B. cinerea mutants constitutively expressing the Pseudomonas putida salicylate hydroxylase NahG, an enzyme that catalyzes salicylic acid degradation, were generated. The NahG-expressing B. cinerea mutants exhibited enhanced in vitro growth on SA-supplemented media, indicating that SA catabolism confers an advantage. In planta, these mutants displayed increased virulence in Arabidopsis thaliana and Phaseolus vulgaris. Notably, the increase in lesion formation was strictly dependent on host SA biosynthesis, as no differences were observed when infecting the SA-deficient Arabidopsis sid2-2 mutant. This result provides evidence that SA degradation increases the virulence of B. cinerea in the interaction with A. thaliana. Genome inspection revealed that the fungus encodes four salicylate hydroxylase-like genes. Analysis of publicly available transcriptomic data from virulence assays across multiple plant hosts revealed that all these genes are expressed during the plant-pathogen interaction, with distinct expression patterns across infection stages and hosts. Together, these observations suggest that B. cinerea may have endogenous mechanisms for SA degradation during host colonization, thereby conferring the capacity to control its accumulation during the infection process. HIGHLIGHTSO_LIBotrytis cinerea expressing the salicylate hydroxylase (SH) ppNahG shows enhanced virulence in Arabidopsis and bean plants. C_LIO_LIEnhanced virulence of NahG-expressing strains depends on host salicylic acid biosynthesis. C_LIO_LIThe Botrytis cinerea genome encodes four SH-like genes. C_LIO_LISH-like genes display distinct expression patterns during infection across different plant hosts. C_LI VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/698134v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@135adorg.highwire.dtl.DTLVardef@16b2947org.highwire.dtl.DTLVardef@62fd40org.highwire.dtl.DTLVardef@e07e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Candidatus Liberibacter asiaticus (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen s biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared to previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB. FundingThis work is supported by USDA National Institute of Food and Agriculture award No. 2020-70029-33197 to W.M and A.L.

Erwinia tracheiphila (Smith) is a recently emerged plant pathogen that causes severe economic losses in cucurbit crops in temperate Eastern North America. E. tracheiphila is xylem restricted, and virulence is thought to be related to Exopolysaccharides (EPS) and biofilm formation, which occlude the passage of sap in xylem vessels and causes systemic wilt. However, the role of EPS and biofilm formation, and their contribution to disease in relation to other virulence loci are unknown. Here, we use deletion mutants to explore the roles of EPS, Hrp Type III secretion system (Hrp T3SS) and Expansin in plant colonization and virulence. Then, we quantify the expression of the genes encoding these factors during infection. Our results show that Exopolysaccharides are essential for E. tracheiphila survival in host plants, while Hrp T3SS and Expansin are dispensable for survival but needed for systemic wilt symptom development. EPS and Hrp T3SS display contrasting expression patterns in the plant, reflecting their relevance in different stages of the infection. Finally, we show that expression of the eps and hrpT3SS operons is downregulated in mildly increased temperatures, suggesting a link between expression of these virulence factors and geographic restriction of E. tracheiphila to temperate regions. Our work highlights how E. tracheiphila virulence is a complex trait where several loci are coordinated during infection. These results further shed light into the relationship between virulence factors and the ecology of this pathosystem, which will be essential for developing sustainable management strategies for this emerging pathogen.

O_LIPhytophthora agathidicida, the causal agent of kauri dieback, secretes RXLR effector proteins to promote host colonisation. One of these, PaRXLR40, was previously shown to suppress immune responses in Nicotiana benthamiana, but its mechanism of action and contribution to virulence remained unclear. C_LIO_LITo investigate PaRXLR40 function, we used comparative approaches in N. benthamiana and Agathis australis (kauri), including RNA interference (RNAi), transient expression assays, confocal microscopy, yeast two-hybrid screens, and infection assays. We also examined host protein interactors and tested mutant variants to evaluate functional domains. C_LIO_LISilencing PaRXLR40 reduced P. agathidicida colonization in N. benthamiana and A. australis. PaRXLR40 interacted with a host BTB/ARM domain protein (ARIA), previously implicated in abscisic acid (ABA) signalling. ARIA suppressed immunity and promoted infection, while interacting with NbSOG1, a DNA damage-associated transcription factor that enhanced resistance when overexpressed. External application of ABA enhanced P. agathidicida infection in both hosts, supporting the hypothesis that PaRXLR40 may hijack host ABA signalling through ARIA to promote susceptibility. C_LIO_LIOur findings show that PaRXLR40 targets ARIA to manipulate host immunity and promote virulence. The interaction between ARIA and SOG1 suggests PaRXLR40 may interfere with host transcriptional reprogramming. PaRXLR40 represents a potential target for future RNAi-based strategies to reduce kauri dieback. C_LI

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, where some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P. pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast-two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRP), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions.

Pantoea agglomerans pv. betae (Pab) induces tumor-like galls in beet and gypsophila, a process mediated by the secretion of effector proteins via Pabs type III secretion system (T3SS). The molecular mechanisms underlying Pab-induced gall formation remain largely unexplored. This study delves into the cellular architecture and transcriptional profile of Pab-mediated galls, comparing host responses to wild-type Pab and a T3SS-inactive mutant, hrcC-. Morphological analysis using scanning electron microscopy and cross-sectional visualization of infected beet leaf tissues revealed that Pab-induced gall-like structures are linked to cell hyperplasia and tissue ruptures, contingent on T3SS activity. Comparative transcriptome analysis of wild-type Pab and hrcC- Pab-infected beet leaves at 12 and 48 hours unveiled significant transcriptional reprogramming, with nearly 2,000 differentially expressed genes at 48 hours post inoculation. Enrichment analyses identified the upregulation of pathways related to signal transduction, defense, carbohydrate metabolism, and cell wall modulation in wild-type Pab-infected leaves compared to controls. Particularly notable was the significant upregulation of numerous genes associated with cell wall loosening by wild-type Pab, suggesting an initial rearrangement of cell wall architecture facilitates gall formation. Furthermore, transcriptome analysis demonstrated that wild-type Pab suppresses the expression of the betalain biosynthetic gene DOPA 4,5-DIOXYGENASE, leading to reduced betalain accumulation in infected tissues compared to the mutant strain. These findings offer fresh insights into the transcriptional and physiological manipulation of host tissue during the early stages of Pab-induced gall formation.

Bacterial fruit blotch (BFB) is a serious disease of melon and watermelon caused by the Gram-negative bacterium Acidovorax citrulli. The strains of the pathogen can be divided into two major genetic groups, I and II. While group I strains are strongly associated with melon, group II strains are more aggressive on watermelon. Like many pathogenic bacteria, A. citrulli secretes a variety of protein effectors to the host cell via the type III secretion system. In the present study, we characterized AopW1, an A. citrulli type III-secreted effector that shares similarity with the actin cytoskeletondisrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. aopW1 is present in group I and II strains, encoding products of 485 amino acids. Although highly conserved in most of the sequence, AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, including 14 amino acid differences between groups. Here we show that group I AopW1 is more toxic to yeast and plant cells than group II AopW1, having a stronger actin filament disruption activity, and increased ability to reduce plant callose deposition. We demonstrate the role of some of these 14 amino acid positions in determining the phenotypic differences between the two versions of the effector. Moreover, cellular analyses revealed that in addition to the interaction with actin filaments, AopW1 is localized to the endoplasmic reticulum, chloroplasts, and early and recycling plant endosomes, with differences observed between the two AopW1 versions. Finally, we show that overexpression of the endosome-associated protein EHD1 that increases cellular recycling, attenuates the toxic effects exerted by AopW1 and increases defence responses. This study provides insights into the HopW1 family of bacterial effectors and their interactions with the plant cell and provides first evidence on the involvement of EHD1 in response to biotic stress.

Genome sequence analyses predicted the presence of effectors in the gram-negative Candidatus Liberibacter asiaticus (CLas) even without the presence of a classical type III secretion system. Since CLas is not culturable, it is not possible to perform traditional gene knockout experiments to determine the role of various effectors in Huanglongbing (HLB) pathogenesis. Therefore, we followed an alternative functional genomics approach to examine the role of the CLas effectors in HLB pathogenesis in general and more specifically in suppressing citrus innate immune response. Here, we focused on the CLas effectors, P235 and Effector 3, to perform the following studies. First, proteomic studies by LC-MS/MS were conducted to screen the putative interacting citrus protein partners of P235 and Effector 3 from the healthy and CLas-infected Hamlin extracts and the most probable candidates were identified based upon their high protein scores from LC-MS/MS. Second, a transgenic tobacco split GFP system was designed for in planta detection of the most probable citrus interacting protein partners of P235 and Effector 3. Third, in vitro and in planta studies were performed to show that each of two effectors interacts with and inhibits the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species (ROS), blocking of bactericidal lipid binding protein (LTP), and induction of premature programmed cell death (PCD), thereby supporting CLas infection and HLB pathogenesis. Finally, an LTP mimic was designed to sequester and block the CLas effector and to rescue the bactericidal activity of LTP.

In Nicotiana benthamiana, expression of the Xanthomonas effector XopQ triggers ROQ1-dependent ETI responses and in parallel accumulation of plastids around the nucleus and the formation of stromules. Both processes were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-mediated plastid reactions are a result of XopQ perception by ROQ1 or a consequence of XopQ virulence activity. We find that N. benthamiana mutants lacking ROQ1, both RNLs (NRG1 and ADR1) or EDS1, fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the RNL double mutant. This analysis aligns XopQ-induced stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast peri-nuclear dynamics, is an integral part of the N. benthamiana ETI response and that both RNL sub-types play a role in this ETI response. One sentence summaryGenetic analysis aligns effector triggered immunity (ETI) induced stromule formation with the ETI signaling cascade but not programmed cell death and questions stromule guided peri-nuclear plastid clustering.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, translocates multiple Transcription Activator-Like Effectors (TALEs) into rice cells. The TALEs localize to the host cell nucleus, where they bind to the DNA in a sequence-specific manner and enhance gene expression to promote disease susceptibility. Xoo strain PXO99A encodes nineteen TALEs, but the host targets of all these TALEs have not been defined. A meta-analysis of rice transcriptome profiles revealed a gene annotated as flavonol synthase/flavanone-3 hydroxylase (henceforth OsS5H/FNS-03g) to be highly induced upon Xoo infection. Further analyses revealed that this gene is induced by PXO99A using TAL9b, a broadly conserved TALE of Xoo. Disruption of tal9b rendered PXO99A less virulent. OsS5H/FNS-03g functionally complemented its Arabidopsis homologue AtDMR6, a well-studied disease susceptibility locus. Biochemical analyses suggested that OsS5H/FNS-03g is a bifunctional protein with Salicylic Acid 5 Hydroxylase (S5H) and Flavone Synthase-I (FNS-I) activities. Further, an exogenous application of apigenin on rice leaves, the flavone that is enzymatically produced by OsS5H/FNS-03g, promoted virulence of PXO99A tal9b-. Overall, our study suggests that OsS5H/FNS-03g is a bifunctional enzyme and its product apigenin is potentially involved in promoting Xoo virulence.

Multicellular organisms require dynamic communication between cells, tissues and organs to integrate responses to external and internal signals. In plants, cell-to-cell communication relies in part on plasmodesmata, which connect adjacent cells and allow the exchange of signals and resources. Upon infection by pathogens, plants act to isolate infected cells from non-infected cells by closing plasmodesmata but pathogens can suppress this defence and maintain plasmodesmata in an open state. To address the question of what a pathogen might gain from keeping plasmodesmata open, we screened effectors from the biotrophic Arabidopsis pathogen Hyaloperonospora arabidopsidis (Hpa) for the ability to move cell-to-cell via plasmodesmata in plant tissues. We quantified the mobility of cytoplasmic effectors and identified six that were hypermobile, i.e., can move further than expected for a protein of that size. Of these, HaRxL77 indirectly modifies plasmodesmatal permeability to facilitate hypermobility and suppresses the flg22-induced ROS burst, suggesting that cell-to-cell mobility of effectors allows defence manipulation ahead of the infection front. Thus, this study provides novel insights into how Hpa exploits plasmodesmata-mediated intercellular connectivity to promote infection, characterising a poorly explored element of plant-pathogen interaction. Author SummaryDuring infection, pathogens secrete effectors into host cells to manipulate them for their benefit. Plant cells are connected via plasmodesmata, and pathogen effectors can use these connections to reach uninfected cells. We asked what a pathogen might gain by having effectors that can travel between cells and thus screened Arabidopsis downy mildew effectors for this capability. We found downy mildew produces many cell-to-cell mobile effectors, and that some of these can move further than we might expect. One hypermobile effector, HaRxL77, can open plasmodesmata to allow this increased mobility, although how it does this is not clear. HaRxL77 interferes with several host defence mechanisms, suggesting that cell- to-cell mobility allows effectors to perturb host defence ahead of the infection front.