Burbank, L. P. | Search bioRxiv

bioRxiv2022

Natural recombination among Type I restriction-modification systems creates diverse genomic methylation patterns among Xylella fastidiosa strains

Xylella fastidiosa is an important bacterial pathogen of plants causing high consequence diseases in agricultural crops around the world. Although as a species X. fastidiosa can infect an extremely broad range of host plants, significant variability exists between strains and subspecies groups in virulence on specific host plant species, and other traits such as growth habits. Natural competence and horizontal gene transfer are believed to occur frequently in X. fastidiosa, and likely influences the evolution of this pathogen. However, some X. fastidiosa strains are extremely difficult or impossible to manipulate genetically using standard transformation techniques. Several restriction-modification systems are encoded in the X. fastidiosa genome, including multiple Type I R-M systems that may influence horizontal gene transfer and recombination. In this study, several conserved Type I R-M systems were compared across 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types. Considerable allelic variation among strains was identified among the single specificity subunit (hsdS) of each Type I R-M system, with a unique hsdS allele profile generally conserved within a monophyletic cluster of strains. Inactivating mutations were identified in Type I R-M systems of specific strains, showing heterogeneity in the complement of functional Type I R-M systems across X. fastidiosa. Genomic DNA methylation patterns were characterized in 20 X. fastidiosa strains and associated with Type I R-M system allele profiles. Overall, this study describes epigenetic modifications in X. fastidiosa associated with functional Type I R-M systems and characterizes the diversity in these systems across X. fastidiosa lineages. ImportanceEconomic impacts on agricultural production due to X. fastidiosa have been severe in the Americas, Europe, and parts of Asia. Despite a long history of research on this pathogen, certain fundamental questions regarding the biology, pathogenicity, and evolution of X. fastidiosa have still not been answered. Wide scale whole genome sequencing has begun to provide a more insight into X. fastidiosa genetic diversity and horizontal gene transfer but the mechanics of genomic recombination in natural settings and extent to which this directly influences bacterial phenotypes such as plant host range are not well understood. Genome methylation is an important factor in horizontal gene transfer and bacterial recombination that has not been comprehensively studied in X. fastidiosa. This study characterizes methylation associated with Type I restriction-modification systems across a wide range of X. fastidiosa strains and lays the groundwork for a better understanding of X. fastidiosa biology and evolution through epigenetics.

microbiology↗

bioRxiv2021

Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant ({Delta}csp1). We observed decreases in cellular aggregation and surface attachment with the {Delta}csp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that {Delta}csp1 had reduced pili compared to the wild type and complemented strains. The {Delta}csp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and {Delta}csp1 revealed changes in expression of several genes important for attachment and biofilm formation in {Delta}csp1. One gene of intertest, pilA1, encodes a type IV pili subunit protein and was up regulated in {Delta}csp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines. X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation. ImportanceXylella fastidiosa is a major threat to the worldwide agriculture industry (1, 2). Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood. There are currently few effective solutions to suppress X. fastidiosa disease development or eliminate bacteria from infected plants(3). Recently, disease epidemics due to X. fastidiosa have greatly expanded(2, 4, 5), exacerbating the need for better disease prevention and control strategies. Our studies show that Csp1 is involved in X. fastidiosa virulence and stress tolerance. Understanding how Csp1 influences pathogenesis and bacteria survival can aide in developing novel pathogen and disease control strategies. We also streamlined a bioinformatics protocol to process and analyze long read Nanopore bacterial RNA-Seq data, which has previously not been reported for X. fastidiosa.

molecular biology↗