Involvement of hrpX and hrpG in the Virulence of Acidovorax citrulli Strain Aac5, Causal Agent of Bacterial Fruit Blotch in Cucurbits.

Xiaoxiao Zhang,Ron Walcott,Linlin Yang,Mei Zhao,Yuwen Yang,Wei Guan,Tingchang Zhao,Jianpei Yan

doi:10.3389/fmicb.2018.00507

Abstract

Acidovorax citrulli causes bacterial fruit blotch, a disease that poses a global threat to watermelon and melon production. Despite its economic importance, relatively little is known about the molecular mechanisms of pathogenicity and virulence of A. citrulli. Like other plant-pathogenic bacteria, A. citrulli relies on a type III secretion system (T3SS) for pathogenicity. On the basis of sequence and operon arrangement analyses, A. citrulli was found to have a class II hrp gene cluster similar to those of Xanthomonas and Ralstonia spp. In the class II hrp cluster, hrpG and hrpX play key roles in the regulation of T3SS effectors. However, little is known about the regulation of the T3SS in A. citrulli. This study aimed to investigate the roles of hrpG and hrpX in A. citrulli pathogenicity. We found that hrpG or hrpX deletion mutants of the A. citrulli group II strain Aac5 had reduced pathogenicity on watermelon seedlings, failed to induce a hypersensitive response in tobacco, and elicited higher levels of reactive oxygen species in Nicotiana benthamiana than the wild-type strain. Additionally, we demonstrated that HrpG activates HrpX in A. citrulli. Moreover, transcription and translation of the type 3-secreted effector (T3E) gene Aac5_2166 were suppressed in hrpG and hrpX mutants. Notably, hrpG and hrpX appeared to modulate biofilm formation. These results suggest that hrpG and hrpX are essential for pathogenicity, regulation of T3Es, and biofilm formation in A. citrulli.

Highlights

Bacterial fruit blotch (BFB), caused by the gram-negative bacterium Acidovorax citrulli (Schaad et al, 1978, 2008; Willems et al, 1992), is a globally occurring destructive disease of cucurbit crop species (Burdman and Walcott, 2012)
To verify whether homologous genes coding HrpG and HrpX exist in strain Aac5, we cloned the sequences from Aac5 and found that they were 100% identical to the sequences of Aave_0445 and Aave_0444 from AAC00-1, named hrpG (GenBank accession no.: MG879251) and hrpX (MG879252), respectively
As hrpG and hrpX were found to be essential for hypersensitive response (HR) induction by A. citrulli, we investigated whether patterntriggered immunity (PTI)-associated Reactive Oxygen Species (ROS) was altered during the A. citrulli–host interaction

Summary

Introduction

Bacterial fruit blotch (BFB), caused by the gram-negative bacterium Acidovorax citrulli (Schaad et al, 1978, 2008; Willems et al, 1992), is a globally occurring destructive disease of cucurbit crop species (Burdman and Walcott, 2012). Gram-negative phytopathogenic bacteria secrete type 3-secreted effectors (T3Es) directly into plant host cells via the T3SS (Block et al, 2008; Van Engelenburg and Palmer, 2010; Nomura et al, 2011; Jiang et al, 2013; Xin et al, 2016) These T3Es may function to overcome pathogen-associated molecular patterntriggered immunity (PTI) and effector-triggered immunity (ETI) (Cui et al, 2013) or promote effector-triggered susceptibility (Jehle et al, 2013). Gram-negative bacterial plant pathogens of the genera Erwinia, Pseudomonas, Ralstonia, and Xanthomonas require a fully active T3SS for pathogenicity, and the recent establishment of an inducing medium has facilitated the study of hrp gene expression and regulation in in-vitro experiments (Yuan and He, 1996; Rossier et al, 1999; Murata et al, 2006; Ancona et al, 2015)

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