A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana.

Chao Zhang,Xuefeng Wang,Weimin Li,Hailin Yan,Peixiu Du,Zongcai Zhu

doi:10.3389/fmicb.2020.594669

Abstract

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.

Highlights

Citrus huanglongbing (HLB), known as citrus greening, is the most devastating worldwide disease of citrus at the moment and greatly damages the citrus industry by shortening lifespan of trees and reducing fruit yield and quality (Bové, 2006; Gottwald, 2010)
These findings indicate that, apart from the Secdependent secretory proteins, Candidatus Liberibacter asiaticus” (CLas) employs non-classically secreted proteins to interfere with the plant defense mechanisms
A BLAST search of the GenBank database revealed that this protein was 100% conserved among the CLas strains with the full genome available (Supplementary Figure 2), but was absent from both Ca. L. africanus” (CLaf) and Ca. L. americanus” (CLam)

Summary

INTRODUCTION

Citrus huanglongbing (HLB), known as citrus greening, is the most devastating worldwide disease of citrus at the moment and greatly damages the citrus industry by shortening lifespan of trees and reducing fruit yield and quality (Bové, 2006; Gottwald, 2010). Two non-classically secreted proteins, CLIBASIA_RS00445 and SC2_gp, were shown to dramatically downregulate the gene expression of RbohB, a gatekeeper of H2O2-mediated defense signaling in Nicotiana tabacum (Jain et al, 2015, 2018), and the former was identified to suppress oxylipin-mediated defense in citrus plants (Jain et al, 2019). These findings indicate that, apart from the Secdependent secretory proteins, CLas employs non-classically secreted proteins to interfere with the plant defense mechanisms. The study revealed an effector of CLas that might contribute to the bacterial pathogenicity

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT