Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1 (original) (raw)
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PLoS Pathogens, 2012
XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv DxopN and Xcv DhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis. Citation: Taylor KW, Kim J-G, Su XB, Aakre CD, Roden JA, et al. (2012) Tomato TFT1 Is Required for PAMP-Triggered Immunity and Mutations that Prevent T3S Effector XopN from Binding to TFT1 Attenuate Xanthomonas Virulence. PLoS Pathog 8(6): e1002768.
Physiological and Molecular Plant Pathology, 2013
Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is an important disease of pomegranate. Most phytopathogenic strains in the Genera Xanthomonas secrete effector proteins by the type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). The T3SS effectors, referred to as Xanthomonas outer proteins (Xops), are known to be key factors required for bacterial growth and colonization in distinct eukaryotic hosts. Xap contains six Xop-effectors, namely XopC2, XopE1, XopL, XopN, XopQ and XopZ. In this study we analyzed xopN, a conserved effector in Xanthomonas, with reference to sequence identity and its role in governing bacterial growth, and virulence. The xopN of Xap shared maximum sequence identity (98.6%) with pathovar citri. Overlapping extension PCR and double crossing over based homologous recombination strategy was employed to generate a xopN null mutant (Xap DxopN) of Xap. A kanamycin gene was used to replace the xopN gene. XopN was required for maximal Xap pathogenicity in its natural host pomegranate. The detailed image analysis on blight lesions revealed 3 fold reduction in watersoaked areas on leaves infiltrated with mutant Xap DxopN compared to that of with wild Xap. The in planta population count of Xap DxopN was reduced approximately 32-fold relative to the wild strain indicating that xopN is required for maximal growth of Xap in pomegranate. In addition, the Xap DxopN mutant induced more callose deposition in infected pomegranate leaves. Taken together, the present study shows that XopN governs Xap growth and modulates cell-wall associated immune response in pomegranate.
2013
Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc. Citation: Guy E, Lautier M, Chabannes M, Roux B, Lauber E, et al. (2013) xopAC-triggered Immunity against Xanthomonas Depends on Arabidopsis Receptor-Like Cytoplasmic Kinase Genes PBL2 and RIPK. PLoS ONE 8(8): e73469.
Journal of mycology and plant pathology, 2019
Pomegranate is a premier export intended crop in India. Of late, bacterial blight disease in pomegranate is causing significant yield loss. The disease causing bacteria pv (Xap) Xanthomonas axonopodis punicae secretes type III effector proteins to suppress the pathogen associated molecular pattern (PAMP)-triggered plant immunity (PTI) and fosters the disease. The role and induction of genes during host-pathogen effector interaction in pomegranate Xap is not known. In this context, an attempt was made to identify the expression profile of ten putative effectors (pathogenicity-related genes) during host-pathogen interaction. These candidate pathogenicity-related genes were identified through analysis, which includes in silico seven genes with putative type III secretion system (XopAK, XopV1, XopP, XopN, XopR, HpaP and HrpB), and one each of a general secretory pathway gene (GspH), a lipase family protein gene (Abhydrolase) and a serine/threonine kinase gene (PKc). The PCR-based amplification confirmed the homology between and analysis of selected candidate effector proteins. Expression analysis in silico in vivo through real-time quantitative PCR showed the induction of candidate pathogenicity-related genes during infection. The XopN, Abhydrolase and PKc gene were highly induced on an interaction between hostpathogen, than pathogen alone. The expression of these effector genes in the host during different time points of infection provided significant insight to deciphering the virulence mechanism of Xap. Unidentified and potentially new virulence factors were reported, which could be subverting host cell resistance processes.
Microbiological Research, 2016
Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease of pomegranate. Xap secretes effector proteins via type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). Previously we reported that XopN, a conserved effector of Xap, modulate in planta bacterial growth, and blight disease. In continuation to that here we report the deletion of XopN from Xap caused higher accumulation of reactive oxygen species (ROS) including H 2 O 2 and O 2 −. We quantitatively assessed the higher accumulation of H 2 O 2 in pomegranate leaves infiltrated with Xap xopN compared to Xap wild-type. We analysed that 1.5 to 3.3 fold increase in transcript expression of ROS and flg22-inducible genes, namely FRK1, GST1, WRKY29, PR1, PR2 and PR5 in Arabidopsis when challenged with Xap xopN; contrary, the up-regulation of all the genes were compromised when challenged with either Xap wild-type or Xap xopN + xopN. Further, we demonstrated the plasma-membrane based localization of XopN protein both in its natural and experimental hosts. All together, the present study suggested that XopN-T3SS effector of Xap gets localized in the plasma membrane and suppresses ROS-mediated early defense responses during blight pathogenesis in pomegranate.
Molecular plant …, 2008
Xanthomonas axonopodis pv. citri (Xac) and Xanthomonas axonopodis pv. aurantifolii pathotype C (Xaa) are responsible for citrus canker disease; however, while Xac causes canker on all citrus varieties, Xaa is restricted to Mexican lime, and in sweet oranges it triggers a defence response. To gain insights into the differential pathogenicity exhibited by Xac and Xaa and to survey the early molecular events leading to canker development, a detailed transcriptional analysis of sweet orange plants infected with the pathogens was performed. Using differential display, suppressed subtractive hybridization and microarrays, we identified changes in transcript levels in approximately 2.0% of thẽ 32 000 citrus genes examined. Genes with altered expression in response to Xac/Xaa surveyed at 6 and 48 h post-infection (hpi) were associated with cell-wall modifications, cell division and expansion, vesicle trafficking, disease resistance, carbon and nitrogen metabolism, and responses to hormones auxin, gibberellin and ethylene. Most of the genes that were commonly modulated by Xac and Xaa were associated with basal defences triggered by pathogen-associated molecular patterns, including those involved in reactive oxygen species production and lignification. Significantly, we detected clear changes in the transcriptional profiles of defence, cell-wall, vesicle trafficking and cell growth-related genes in Xac-infected leaves between 6 and 48 hpi. This is consistent with the notion that Xac suppresses host defences early during infection and simultaneously changes the physiological status of the host cells, reprogramming them for division and growth. Notably, brefeldin A, an inhibitor of vesicle trafficking, retarded canker development. In contrast, Xaa triggered a mitogen-activated protein kinase signalling pathway involving WRKY and ethylene-responsive transcriptional factors known to activate downstream defence genes.
Proceedings of the National Academy of Sciences, 2004
The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resulted in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the
Molecular plant pathology, 2017
Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 has 17 predicted T3Es belonging to the Xanthomonas outer protein class (Xop). This work aimed at characterizing nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1, and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in PAMP-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI) using heterologous systems. AvrBs2, XopR, and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs...
Identification of a host 14-3-3 protein that interacts with Xanthomonas effector AvrRxv
Physiological and Molecular Plant Pathology, 2008
AvrRxv is a member of a family of pathogen effectors present in pathogens of both plant and mammalian species. Xanthomonas campestris pv. vesicatoria strains carrying AvrRxv induce a hypersensitive response (HR) in the tomato cultivar Hawaii 7998. Using a yeast two-hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatography. Using mutagenesis, we identified a 14-3-3-binding domain in AvrRxv and demonstrated that a mutant in that domain showed concomitant loss of interaction with ARI1 and HR-inducing activity in tomato. These results demonstrate that the AvrRxv bacterial effector recruits 14-3-3 proteins for its function within host cells. AvrRxv homologues YopP and YopJ from Yersinia do not have AvrRxv-specific HR-inducing activity when delivered into tomato host cells by Agrobacterium. Although YopP itself cannot induce HR, its C-terminal domain containing the catalytic residues can replace that of AvrRxv in an AvrRxv-YopP chimera for HR-inducing activity. Phylogenetic analysis indicates that the sequences encoding the C-termini of family members are evolving independently from those encoding the N-termini. Our results support a model in which there are three functional domains in proteins of the family: translocation, interaction, and catalytic.