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ARS Home » Pacific West Area » Parlier, California » San Joaquin Valley Agricultural Sciences Center » Crop Diseases, Pests and Genetics Research » Research » Publications at this Location » Publication #414667
Research Project: Development of Applied Management Systems for Diseases of Perennial Crops with Emphasis on Vector-Borne Pathogens of Grapevine and Citrus

Location: Crop Diseases, Pests and Genetics Research

Title: In planta transposon sequencing for virulence gene identification in Xylella fastidiosa

Author
item Burbank, Lindsey
item DEYETT, ELIZABETH - University Of California, Riverside
item HER, NANCY - University Of California, Riverside
item Magdaleno, Mayra
item Helm-Rodriguez, Sydney
item ROLSHAUSEN, PHILLIPE - University Of California, Riverside
item ROPER, CAROLINE - University Of California, Riverside

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Xylella fastidiosa causes significant economic damage in multiple agricultural crops. Globally, disease epidemics in citrus, grapes, almonds, and olives launched widespread efforts in pathogen surveillance, and quarantine restrictions on plant commodities. Research efforts on Xylella fastidiosa biology and pathogenesis depend on development and adaptation of protocols and techniques in bacterial genetics. Transposon-sequencing (Tnseq) is a method used to identify essential bacterial genes during host infection by high-throughput sequencing of randomly-generated mutants. In this study, a Tnseq protocol was adapted for identifying essential genes in Xylella fastidiosa during plant infection. Several Xylella fastidiosa genes were identified as essential for infection that had not previously been characterized, broadening existing knowledge on how Xylella fastidiosa causes disease. Expansion of available tools for research, such as adapting Tnseq for Xylella fastidiosa plant experiments, will facilitate identification of novel disease control targets for this pathogen.

Technical Abstract: In bacterial genetics, large-scale screening approaches such as sequencing transposon mutant pools can be highly effective for identifying and characterizing genes with unknown functions. In the plant pathogen Xylella fastidiosa, this approach is challenging due to the fastidious nature of this bacterial species and its niche-specific growth in the plant xylem tissue. The purpose of this study was to explore feasibility of transposon sequencing (Tnseq) for identification of novel virulence genes in X. fastidiosa. Predicted essential genes were compared after X. fastidiosa growth in vitro, in grapevine, and in almond. After growth in planta, several gene categories were highlighted as essential including hemagglutinins, tRNAs, toxin-antitoxin systems, and prophage genes. Additionally, several hypothetical protein genes were identified that may be good candidates for future characterization. Three predicted essential genes (XfasM23_0359, XfasM23_0360, XfasM23_0972) were chosen for further characterization by making targeted deletion mutants. Deletion mutants exhibited reduced disease in grapevines, but normal growth and aggregation phenotypes in vitro. Overall, the Tnseq approach has some practical limitations due to the nature of the X. fastidiosa pathosystem, but is still able to identify genes important for disease in plants. Future improvements and variations of this Tnseq protocol for X. fastidiosa such as using different sampling methods, pathogen strains, or host susceptibility levels will facilitate research on disease mechanisms and novel pathogenicity determinants.