<em>Pseudomonas syringae</em> Effector Avirulence Protein E Localizes to the Host Plasma Membrane and Down-Regulates the Expression of the <em>NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE13</em> Gene Required for Antibacterial Immunity in Arabidopsis^{<a href="#fn1" rid="fn1" class=" fn">1</a>,}^{<a href="#fn2" rid="fn2" class=" fn">[OPEN]</a>}

Abstract

As a common mechanism of pathogenesis, many animal and plant pathogenic bacteria utilize the type III secretion system (T3SS) to deliver effector proteins into the host cell (Galán and Collmer, 1999; He et al., 2004). For the past decade, Pseudomonas syringae pv tomato (Pst) strain DC3000 has been employed as a model pathogen to define the virulence functions of type III secretion system effectors (T3Es). Study of T3Es has led to significant advances not only in the understanding of the fundamental mechanisms underlying bacterial pathogenesis but also in the discovery and characterization of functions of relevant plant genes (Block and Alfano, 2011; Dou and Zhou, 2012; Xin and He, 2013). However, despite much progress in this area, the virulence functions of most T3Es from Pst DC3000 and other plant pathogenic bacteria remain undefined, illustrating substantial potential in using pathogen virulence factors as probes in the characterization of the functions of plant genes.

Among the most crucial T3Es of plant pathogenic bacteria is the Avirulence Protein E (AvrE) family of effectors, of which AvrE from Pst DC3000 is the founding member (Lorang and Keen, 1995; Degrave et al., 2015). The AvrE effector family is present in diverse plant pathogenic bacteria that belong to the genera Pseudomonas, Pantoea, Erwinia, Dickeya, and Pectobacterium. Importantly, mutation of avrE orthologs has been shown to cause a dramatic decrease in the virulence of a number of bacteria (Gaudriault et al., 1997; Bogdanove et al., 1998; Frederick et al., 2001; DebRoy et al., 2004; Boureau et al., 2006). In P. syringae, AvrE is functionally redundant to another effector protein, Hypersensitive response and pathogenicity-dependent Outer Protein M1 (HopM1; Alfano et al., 2000; DebRoy et al., 2004; Badel et al., 2006). Mutation of either avrE or hopM1 alone does not strongly affect Pst DC3000 virulence, but the avrE hopM1 double mutant is severely impaired in virulence (Badel et al., 2006). Because of the crucial virulence role of AvrE family effectors and the wide distribution of this effector family in diverse plant pathogenic bacteria, an understanding of the virulence functions of AvrE family effectors is expected to have a substantial impact on our understanding of bacterial diseases in plants. Similarly, because the virulence functions of AvrE family effectors are not understood at the molecular level, study of AvrE family effectors may lead to new insights into the functions of relevant plant genes.

For technical reasons, AvrE family effectors have been very challenging to study due to their extremely large size (approximately 200 kD) and high toxicity to plant and yeast (Saccharomyces cerevisiae) cells. Nevertheless, some progress has been made in the characterization of AvrE family effectors. For example, Ham et al. (2008, 2009) identified two sequence motifs in AvrE family effectors: the WxxxE motif (where x stands for any amino acid other than Trp or Glu) at the N-terminal one-half and a putative endoplasmic reticulum membrane retention/retrieval-like signal (ERMRS) at the C terminus. Some WxxxE effectors from human pathogenic bacteria function as guanine nucleotide-exchange factors to activate small GTPases. In these effectors, the WxxxE motif appears to play a structural role in properly positioning the catalytic loop and is required for the guanine nucleotide-exchange factor activity (Alto et al., 2006; Ohlson et al., 2008; Huang et al., 2009c). However, whether the WxxxE motif in the AvrE family has a similar or a different structural function has yet to be determined. Similarly, it is not known whether the C-terminal ERMRS motif indeed targets AvrE family effectors to the endoplasmic reticulum (ER) in the plant cell.

All AvrE family effectors examined, including AvrE from Pst DC3000, Water-Soaking E (WtsE) from Pantoea stewartii, Disease-specific protein A (DspA)/E from Erwinia amylovora, and DspE from Pectobacterium carotovorum, are strong inducers of water soaking and/or cell death when expressed in host or nonhost plants (Frederick et al., 2001; Boureau et al., 2006; Ham et al., 2006, 2008, 2009; Degrave et al., 2008; Kim et al., 2011; Hogan et al., 2013). Conversely, deletion of avrE family genes delays or abolishes this ability (Bogdanove et al., 1998; Frederick et al., 2001; Badel et al., 2006; Ham et al., 2006). In addition, AvrE family effectors have been shown to suppress plant defense responses, such as callose deposition and expression of the defense gene PATHOGENESIS RELATED1 (DebRoy et al., 2004; Boureau et al., 2006; Ham et al., 2008, 2009), and DspA/E was reported to interact with several putative receptor kinases from apple (Malus domestica; Meng et al., 2006). Intererstingly, DspA/E affects actin dynamics and vesicle trafficking in yeast (Siamer et al., 2011), and a genetic screen identified sphingolipid intermediates and protein phosphatase 2A regulatory subunits important for its toxicity in yeast (Siamer et al., 2014). Transcriptional profiling of the host maize (Zea mays) plants shows that WtsE induces genes involved in secondary metabolism and the suppression of genes involved in photosynthesis (Asselin et al., 2015). In the nonhost plant Arabidopsis (Arabidopsis thaliana), transgenic expression of E. amylovora DspA/E results in the induction of a large suite of salicylic acid (SA)-dependent defense genes (Degrave et al., 2013). However, overall, our understanding of AvrE family effectors remains fragmentary. In particular, we do not know their sites of action in the host cell or the specific host genes that are required for their virulence functions, presenting a significant roadblock to our general understanding of bacterial pathogenesis in plants.

In this study, we initiated efforts to define (1) the subcellular localization of AvrE inside the plant cell and (2) the host gene expression associated with the virulence function of AvrE in the host plant Arabidopsis. These experiments led to the findings that (1) AvrE, although containing no known plasma membrane (PM)-targeting signal, is localized to the host PM and PM-associated vesicle-like structures, (2) AvrE contains two physically interacting domains with the N-terminal portion containing a PM-targeting signal, and (3) importantly, the virulence function of AvrE is linked to down-regulation of the expression of a specific member of the NONRACE-SPECIFIC DISEASE RESISTANCE1/HARPIN-INDUCED1-LIKE (NHL) gene family, NHL13, which we found to be required for antibacterial immunity in Arabidopsis. Together, these results defined the site of action of one of the most important bacterial virulence proteins in plants and the antibacterial immunity function of the NHL13 gene.

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