Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine‐learning approach

The Gram‐negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85‐10 strain by various experimental and computational techniques. To identify additional Xcv 85‐10 effectors, we applied a genome‐wide machine‐learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)‐dependent expression, 5′ regulatory sequences, amino acid composition bias and GC content. Thirty‐six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)‐inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild‐type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild‐type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

Implication of a pepper h-type thioredoxin in type I- and II-nonhost resistance to Xanthomonas axonopodis

Thioredoxins (TRXs) are distributed ubiquitously in prokaryotic and eukaryotic organisms. Plants have the most complex forms of TRXs. The functional roles of such TRXs have been studied in abiotic stress but their roles in plant defense responses against biotic stresses have been less well studied. Here, we identified an h-type TRX gene from pepper, CaTRXh1, and characterized its possible effect on Type II nonhost resistance, which entails localized programmed cell death in response to nonhost pathogens. Peptide sequences of CaTRXh1 showed a high degree of similarity with TRXhs from tobacco and Arabidopsis thaliana. Southern blot analyses revealed that CaTRXh1 was present as a single copy in the pepper genome. Intriguingly, leaf infiltration by Xanthomonas axonopodis pv. glycines 8ra, eliciting a visible type II nonhost hypersensitive response (HR), and its type III secretion-system null mutant 8–13, eliciting a type I nonhost non-HR, both induced CaTRXh1 at a level similar to that of pathogenesis-related protein 4, an HR marker gene in pepper. More surprisingly, expression of CaTRXh1 was significantly increased when X. axonopodis pv. vesicatoria race 3 infiltrated the leaf of a pepper cultivar containing a resistance gene, but not with infiltration of a susceptible pepper cultivar. Taken together, our study suggests that the expression of CaTRXh1 has a critical role in HR-mediated active defense responses in pepper. GenBank accession number: EF371503.     

Xv4-vrxv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and wild tomato relative Lycopersicon pennellii

Strains of tomato race 3 (T3) of Xanthomonas campestris pv. vesicatoria elicit a hypersensitive response (HR) in leaves of Lycopersicon pennellii LA716. Genetic segregation of the resistance exhibited ratios near 3:1 in F2 populations, which confirmed that a single dominant gene controlled the inheritance of this trait. With the aid of a collection of introgression lines, restriction fragment length polymorphism, and cleaved amplified polymorphic sequence markers, the resistance locus was located on chromosome 3 between TG599 and TG134. An avirulence gene named avrXv4 was also isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp. The predicted AvrXv4 protein exhibits high similarity to members of an emerging new family of bacterial proteins from plant and mammalian pathogens comprising AvrRxv, AvrBsT, YopJ, YopP, AvrA, and YL40.     

Type III effector proteins from the plant pathogen Xanthomonas and their role in the interaction with the host plant

Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria and most other Gram-negative bacterial plant pathogens largely depends on a type III secretion (TTS) system which is encoded by hypersensitive response and pathogenicity (hrp) genes. These genes are induced in the plant and are essential for the bacterium to be virulent in susceptible hosts and for the induction of the hypersensitive response (HR) in resistant host and non-host plants. The TTS machinery secretes proteins into the extracellular milieu and effector proteins into the plant cell cytosol. In the plant, the effectors presumably interfere with cellular processes to the benefit of the pathogen or have an avirulence activity that betrays the bacterium to the plant surveillance system. Type III effectors were identified by their avirulence activity, co-regulation with the TTS system and homology to known effectors. A number of effector proteins are members of families, e.g., the AvrBs3 family in Xanthomonas. AvrBs3 localizes to the nucleus of the plant cell where it modulates plant gene expression. Another family that is also present in Xanthomonas is the YopJ/AvrRxv family. The latter proteins appear to act as SUMO cysteine proteases in the host. Here, we will present an overview about the regulation of the TTS system and its substrates and discuss the function of the AvrRxv and AvrBs3 family members in more detail.     

Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

Characterization of AvrBs3-like effectors from a Brassicaceae pathogen reveals virulence and avirulence activities and a protein with a novel repeat architecture

Xanthomonas campestris pv. armoraciae strain 5 is a Brassicaceae pathogen that expresses three members of the highly related avrBs3 gene family of type III effectors. Here, we report on the isolation and characterization of these genes, designated hax2, hax3, and hax4 (homolog of avrBs3 in Xanthomonas). All three Hax proteins are translocated from Xanthomonas spp. into the plant cell via the type III secretion system. Hax3 and Hax4 show the typical structure of AvrBs3-like effectors and contain a repetitive region in their central part consisting of 34-amino-acid (aa) repeats. By contrast, the Hax2 repeat region is composed of 35-aa repeats that are characterized by an additional proline residue. Hax2, Hax3, and Hax4 contain 21.5, 11.5, and 14.5 repeats, respectively. Genetic studies revealed an additive effect of hax2, hax3, and hax4 on disease symptom formation of X. campestris pv. armoraciae strain 5 on radish. The contribution of individual genes to the aggressiveness of strain 5 is quantitatively different, with hax2 showing the strongest effect on the development of chlorosis and necrosis. In addition, hax3 and hax4, but not hax2, have a Bs4-dependent avirulence activity in tomato and in transgenic Nicotiana benthamiana expressing the Bs4 resistance gene.     

Liposome-bilirubin interaction: a novel strategy to eliminate bilirubin from systemic circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin-liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome-mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

GYF domain proteomics reveals interaction sites in known and novel target proteins

GYF domains are conserved eukaryotic adaptor domains that recognize proline-rich sequences. Although the structure and function of the prototypic GYF domain from the human CD2BP2 protein have been characterized in detail, very little is known about GYF domains from other proteins and species. Here we describe the binding properties of four GYF domains of various origins. Phage display in combination with SPOT analysis revealed the PPG(F/I/L/M/V) motif as a general recognition signature. Based on these results, the proteomes of human, yeast, and Arabidopsis thaliana were searched for potential interaction sites. Binding of several candidate proteins was confirmed by pull-down experiments or yeast two-hybrid analysis. The binding epitope of the GYF domain from the yeast SMY2 protein was mapped by NMR spectroscopy and led to a structural model that accounts for the different binding properties of SMY2-type GYF domains and the CD2BP2-GYF domain.     

Marker-assisted development and characterization of near-isogenic lines carrying the Rx4 gene for hypersensitive resistance to Xanthomonas euvesicatoria pv. perforans race T3 in tomato

Molecular Breeding - Stigma exsertion is a plant trait wherein stigmas emerge from flower buds before anthesis. It is a key determinant of the ornamental kale mating system, greatly increases the...     

Identification of the substrates and interaction proteins of aurora kinases from a protein-protein interaction model

The increasing use of high-throughput and large-scale bioinformatics-based studies has generated a massive amount of data stored in a number of different databases. The major need now is to explore this disparate data to find biologically relevant interactions and pathways. Thus, in the post-genomic era, there is clearly a need for the development of algorithms that can accurately predict novel protein-protein interaction networks in silico. The evolutionarily conserved Aurora family kinases have been chosen as a model for the development of a method to identify novel biological networks by a comparison of human and various model organisms. Our search methodology was designed to predict and prioritize molecular targets for Aurora family kinases, so that only the most promising are subjected to empirical testing. Four potential Aurora substrates and/or interacting proteins, TACC3, survivin, Hec1, and hsNuf2, were identified and empirically validated. Together, these results justify the timely implementation of in silico biology in routine wet-lab studies and have also allowed the application of a new approach to the elucidation of protein function in the post-genomic era.