Temporal Quantitative Changes in the Resistant and Susceptible Wheat Leaf Apoplastic Proteome During Infection by Wheat Leaf Rust (Puccinia triticina).

Wheat leaf rust caused by the pathogenic fungus, Puccinia triticina, is a serious threat to bread wheat and durum production in many areas of the world. This plant-pathogen interaction has been studied extensively at the molecular genetics level however, proteomics data are still relatively scarce. The present study investigated temporal changes in the abundance of the apoplastic fluid proteome of resistant and susceptible wheat leaves infected with P. triticina race-1, using a label-free LC-MS-based approach. In general, there was very little difference between inoculated and control apoplastic proteomes in either host, until haustoria had become well established in the susceptible host, although the resistant host responds to pathogen challenge sooner. In the earlier samplings (up to 72 h after inoculation) there were just 46 host proteins with significantly changing abundance, and pathogen proteins were detected only rarely and not reproducibly. This is consistent with the biotrophic lifestyle of P. triticina, where the invading pathogen initially causes little tissue damage or host cell death, which occur only later during the infection cycle. The majority of the host proteins with altered abundance up to 72 h post-inoculation were pathogen-response-related, including peroxidases, chitinases, ß-1-3-endo-glucanases, and other PR proteins. Five days after inoculation with the susceptible apoplasm it was possible to detect 150 P. triticina proteins and 117 host proteins which had significantly increased in abundance as well as 33 host proteins which had significantly decreased in abundance. The latter represents potential targets of pathogen effectors and included enzymes which could damage the invader. The pathogen-expressed proteins-seen most abundantly in the incompatible interaction-were mostly uncharacterized proteins however, many of their functions could be inferred through homology-matching with pBLAST. Pathogen proteins also included several candidate effector proteins, some novel, and some which have been reported previously. All MS data have been deposited in the PRIDE archive (www.ebi.ac.uk/pride/archive/) under Project PXD012586.

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum.

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys325 ) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys325 to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.

Serological assessment of synthetic peptides of Campylobacter jejuni NCTC11168 FlaA protein using antibodies against multiple serotypes.

The flagellum of Campylobacter jejuni is not only responsible for initiating colonization of the gastrointestinal tract of host animals but is also a major antigen that induces protective immune responses. However, protection is limited to the homologous strain and the ability to protect against multiple serotypes has yet to be determined. In this study, we have shown that FlaA is an immunodominant protein on NCTC11168 CJ1 flagella and we mapped the immunoreactive epitopes on the protein by probing a series of overlapping synthetic peptides spanning the entire sequence with sera against multiple C. jejuni serotypes. Amino acid residues 176-205 (P8), 376-405 (P16) and 501-530 (P21) were immunodominant and cross-reactive. The mucosal IgA in the intestinal secretions of CJ1-infected birds reacted significantly with peptides P16 and P21 indicating that the specificity of the mucosal response is different from the systemic response. Antisera raised against formalin-killed CJ1 cells and purified flagellin showed positive reactivity with a subset of peptides identified by antisera against live C. jejuni. This study provides insight into the specificity of the host immune responses to the FlaA protein of C. jejuni and suggests that these sequences merit further testing for their immunogenicity and potential as subunit vaccine candidates for multiple serotypes.

Influence of Campylobacter jejuni fliA, rpoN and flgK genes on colonization of the chicken gut.

Campylobacter jejuni, a commensal Gram-negative motile bacterium commonly found in chickens is a frequent cause of human gastrointestinal infections. The polar flagellum of C. jejuni is an important virulence and colonization factor, providing motility to the cell as well as a type III secretion function. The flagellar biosynthesis genes fliA (sigma28) and rpoN (sigma54) of C. jejuni regulate a large number of genes involved in motility, protein secretion and invasion, which have been shown to be important factors for the virulence of this organism. To understand the role of the flagellar sigma factors, sigma28 and sigma54, in regulating colonization of the chicken intestinal tract, we assessed fliA and rpoN mutants of C. jejuni NCTC11168 for their ability to secrete Cia proteins and to adhere to and invade Hela cells. The mutants were also tested for their in vivo colonization potential in a chicken model with two different challenge doses. The fliA mutant showed reduced motility (25% that of the wild type) but secreted Cia proteins, yet it did not colonize the chicken cecum. The rpoN mutant cells lacked the spiral shape of C. jejuni and motility was reduced to 10% of the wild-type. The rpoN mutant did not secrete any Cia proteins but RT-PCR analysis showed the presence of ciaB mRNA, indicating that ciaB gene expression was independent of sigma54. Not surprisingly, the colonization defects of both fliA and rpoN mutants were more severe than the flgK mutant. We also demonstrated that FlgK, the hook filament junction protein of C. jejuni, is required for assembly of the flagellar secretory apparatus and an flgK mutant of C. jejuni expressing only the hook showed diminished motility and was completely attenuated for cecal colonization in chickens.

Effect of cytolethal distending toxin of Campylobacter jejuni on adhesion and internalization in cultured cells and in colonization of the chicken gut.

Campylobacter jejuni produces cytolethal distending toxin (CDT) that causes host cells to arrest during their cell cycle and that is involved in the pathogenesis of inflammatory diarrhea in humans. To assess the role of CDT in adherence and invasion of different cultured host cells (HeLa and HD-11) and in colonization of the chicken intestine, the genes of C jejuni NCTC11168 encoding the toxin subunits (cdtA, cdtB, and cdtC) were inactivated by insertional mutagenesis. No significant difference was found in adhesion of the wild-type C. jejuni and the isogenic mutants to HeLa and HD-11 cells. All of the mutants exhibited a decrease (>10-fold) in the ability to invade HeLa cells, but no significant difference was noticed for HD-11 cells. The ability of mutants to colonize birds either directly or by horizontal transfer was unchanged. These data indicated that although the production of cytotoxin does not play a role in the adherence to either human or avian cells, it may play a role in the invasion, survival, or both of C. jejuni in human cells, which are more susceptible to C. jejuni internalization. The CDT also does not seem to play a role in the colonization of poultry.