Browsing by Author "Nyembe, Nompumelelo Philile Praiseworth"

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    Development of a reporter system for the analysis of xylophilus ampelinus type III secreted effectors
    (University of the Western Cape, 2014) Nyembe, Nompumelelo Philile Praiseworth; Ndimba, Bongani K.; Petersen, Yolanda
    Xylophilus ampelinus, the causal agent of bacterial blight and canker of grapevines, has long been a threat to the table grape industry in the Western Cape, leading to severe economic losses due to the reduced productivity and shortened lifespan of infected grapevines. Very little is known about the genetic makeup of the organism, especially with regard to the factors that contribute to its pathogenicity. Generally, bacterial pathogens directly inject the effector proteins into host cells via Type III secretion system (T3SS). In the attempts to identify and characterize the T3 secreted effectors, different reporter plasmid systems have been used to study the secretion and translocation mechanisms the effectors employ during pathogenicity. The aim of the study was to generate a T3 reporter plasmid system for X. ampelinus that will allow the identification and classification of potential pathogenicity factors as members of the Type III secretion class of effectors. First, the avrBs1 family genes avrBs1 and avrA were identified and characterized. The two avirulence genes induced HR on Nicotiana tabacum leaves. Due to the relatedness of the X. ampelinus avr sequences to those of xanthomonads, and the fact that Xanthomonas avrBs1 has been successfully used in a number T3 effector studies, it was decided to construct an X. ampelinus T3 effector reporter vector based on the avrBs1 gene. The minimal segment of the X. ampelinus AvrBs1 protein C-terminus, sufficient for recognition inside host cells and also responsible for HR-induction was identified and characterized using Agrobacterium-mediated transient expression. The AvrBs157-413 HR-inducing domain was cloned in-frame with the 3x FLAG epitope, into a broad-host range vector. To test the reporter vector, the full length avrBs1 sequences of X. ampelinus and Xanthomonas campestris pv. campestris were cloned ahead of the 3x FLAG epitope and the constructs were transferred into XaΔavrBs1 knockout mutant to test for protein secretion. Furthermore, the reporter construct was tested for Type III protein translocation on Bs1 resistant pepper cultivar STAR 6657. Optimization of protein secretion and translocation assays is however required for the improved results. This might include the application of an alternative protein tag to identify candidate X. ampelinus T3SS effectors.
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    Proteomic and functional characterisation of biofilm formation in Xylophilus ampelinus
    (University of the Western Cape, 2019) Nyembe, Nompumelelo Philile Praiseworth; Ludidi, Ndiko
    Xylophilus ampelinus, the causal agent of bacterial blight of grapevine, inhabits the vascular tissues of infected plants where the cells appear to form assemblages resembling biofilms. Bacterial blight of grapevine affects vine-growing areas in Europe and parts of the Mediterranean, Japan, and South Africa. Very little is known about the genetics and pathogenicity factors of the bacterium. The aim of this study was to characterize the biofilm formation process of X. ampelinus through the analysis of its biofilm proteome and functional characterization of the type IV pili (T4P). Biofilm formation allows the bacteria to grow on surfaces, produce and respond to signals, and alter their phenotypes through gene expression to promote adaptation and virulence. To characterize the biofilms formed by X. ampelinus, an in vitro biofilm formation assay was used to identify the stages of biofilm formation. The four major stages of biofilm formation identified, namely attachment, initial biofilm (micro-colony) formation, biofilm maturation, and dispersal, occurred at day three, fiveseven, ten, and fifteen, respectively. When comparing the protein profiles from the initial and mature biofilm stages against the planktonic culture protein profile, 254 spots - showing a twofold change - were considered to be differentially expressed. Out of 99 differentially expressed proteins selected for identification, fifty-nine protein spots yielded 82 protein identities that were assigned to seven functional categories including cellular processes, environmental adaptation, environmental information processing, genetic information processing, metabolism, membrane transport, and proteins of unknown function. Most proteins involved in genetic information processing were induced in abundance during both stages of biofilm formation whereas most proteins involved in carbohydrate, amino acid, and energy metabolism were reduced. Among the proteins identified, a PAS domain S-box-containing protein/diguanylate cyclase (GGDEF)-like protein was induced during the initial biofilm stage, while an indication that X. ampelinus biofilm formation process and regulation require the secondary messenger cyclic di-GMP, this was further confirmed by detection of proteins involved in two of the c-di-GMP-targeted pathways required for cellulose and poly-N-acetylglucosamine (PNAG) biosynthesis. Bioinformatic analysis also identified five proteins with similarity to the proteins encoded in the rpf (regulation of pathogenicity factors) cluster of phytobacteria, which are required for DSF quorum-sensing signal synthesis and perception. Furthermore, the role of T4P in attachment and biofilm formation was identified through the functional characterization of six X. ampelinus genes, namely pilA, pilB, http://etd.uwc.ac.za/ II pilC, pilD, pilQ, and pilU. Scanning transmission electron microscopy (STEM) showed that pilA, pilB, pilC, pilD, and pilQ genes are indispensable in the biogenesis and expression of T4P, while the twitching motility gene, pilU, was not required for T4P expression. All T4P mutants showed defects in the ability to twitch on agar surface under inducing conditions. Consequently, T4P mutants (pilA, pilB, pilC, and pilD) were impaired in their ability to form biofilms in vitro while pilQ and pilU mutants were not affected. In planta studies of the mutant and complemented T4P strains showed that pilA, pilB, pilC, pilD, and pilQ deficient mutant strains were unable to form biofilms both at the inoculation point (IP) and at distal parts from the IP, indicating that T4P are required for host-surface colonization and establishment of bacteria inside the host plant. The pilU mutant retained the wild-type phenotype in biofilm formation both in vitro and at the point of inoculation in planta, however, the same mutant failed to form micro-colonies and biofilms at distal parts from the IP indicating that the translocation of the cells across surfaces requires the retraction function of the T4P that drives twitching motility. The observed role of T4P in biofilm development inside plants and the reduced virulence exhibited by all the T4P mutants indicate that T4P is required for in planta biofilm formation and surface colonization through twitching motility and that T4P is required for the virulence of X. ampelinus in its host, grapevine. Together, the biofilm proteome analysis and functional characterization of X. ampelinus T4P provide new insights into biofilm formation, its regulation and contribution to the virulence of X. ampelinus.