Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

Shujian Zhang,Dean W Gabriel,Donald L Hopkins,Laura A Fleites,Patricia A Rayside,Pranjib K Chakrabarty

doi:10.1371/journal.pone.0133796

Abstract

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

Highlights

Xylella fastidiosa (X. fastidiosa) is a fastidious, insect-vectored, plant xylem-limited bacterial pathogen that causes a variety of diseases in a wide range of plant species [1]
Suspected pathogenicity genes found in Temecula1 that appeared to be missing from EB92-1 [19] were further investigated by designing PCR primers internal to and outside of the coding sequences (CDSs) of PD1703 (LipA), PD0956 (Serine protease), PD0915 (Zot), PD0928 (Zot) and PD0986 (S1 Table)
Two sets of primers were designed to attempt detect these genes in EB92-1; one set was designed internal to the CDS of each gene (92% of predicted EB92-1 proteins had more than 99% identity with Temecula1 proteins [19]) and at least one additional set was designed external to the CDS in Temecula1

Summary

Introduction

Xylella fastidiosa (X. fastidiosa) is a fastidious, insect-vectored, plant xylem-limited bacterial pathogen that causes a variety of diseases in a wide range of plant species [1]. Three New Xylella Pathogenicity Effectors damaging X. fastidiosa diseases are Pierce’s disease (PD) of Vitis viniferea grapevines, leaf scorch of almonds, and citrus variegated chlorosis (CVC). X. fastidiosa cells multiply and spread widely from the site of infection to colonize the xylem of susceptible grapes. The symptoms of PD include leaf scorch and xylem occlusion, resulting in the eventual death of the vine [1]. Among the earliest symptoms caused by X. fastidiosa on grape is a characteristic leaf scorch that, similar to symptoms of water stress, often occurs in bands, which is suggestive of either toxin or pathogenicity effector activity [2,3,4]

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