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

Abstract

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.