NopL, an Effector Protein ofRhizobiumsp. NGR234, Thwarts Activation of Plant Defense Reactions

Abstract

Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and ␤-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.Plants have evolved many defenses against invading pathogens. Among them are preformed antimicrobial compounds, as well as inducible defense proteins, the so-called pathogenesis-related (PR) proteins (van Loon, 1997). It has been shown, for example, that pathogen-induced chitinases (EC 3.2.1.14) and ␤-1,3-glucanases (EC 3.2.1.39) synergistically lyse fungal cell walls (Mauch et al., 1988). Specific recognition of pathogens often induces a hypersensitive response, which is characterized by an oxidative burst and localized death of the host cells. In most cases, the induction of a hypersensitive response arrests invasion by the pathogen (Dangl and Jones, 2001).Virulence in gram-negative bacteria often depends on proteins injected into eukaryotic cells. Some bacteria elaborate a specialized protein secretion apparatus, the type III secretion system (TTSS). The TTSS exports a set of proteins from the bacteria, some of which are delivered directly into the eukaryotic cells-a process called translocation (Cornelis and van Gijsegem, 2000;Plano et al., 2001). Most translocated type III effectors act on the cytoskeleton or interfere with intracellular signaling cascades of the host cell. Yersinia pestis, for example, injects at least six type III effectors into host cells, where they thwart the signaling machinery of the immune system (Cornelis, 2002). Plant pathogens such as Pseudomonas syringae and Xanthomonas campestris also use TTSSs (Kjemtrup et al., 2000; Lahaye and Bonas, 2001). Mounting evidence suggests that type III effectors translocated into plant cells (Casper-Lindley et al., 2002;Szurek et al., 2002) act as virulence factors in susceptible hosts. One likely function of type III effectors is ...

Functional Analysis of the Type 3 Effector Nodulation Outer Protein L (NopL) from Rhizobium sp. NGR234

“…Previous studies showed that transgenic plant cells expressing the type 3 effector gene nopL of NGR234 showed effects on expression of defense genes, suggesting that NopL can suppress the innate immunity of the plant (24). These findings were consistent with the observation that a nopL mutant of NGR234 (strain NGR⍀nopL) induced fewer nodules on the legume Flemingia congesta (22).…”

“…These findings were consistent with the observation that a nopL mutant of NGR234 (strain NGR⍀nopL) induced fewer nodules on the legume Flemingia congesta (22). Experiments with recombinant NopL protein expressed in Escherichia coli revealed that NopL could be phosphorylated at unknown sites by nondefined protein kinases from crude plant protein extracts (23,24). Phosphorylation of NopL in vitro was reduced by addition of PD98059, a MAPK kinase inhibitor (23).…”

“…Treatment of NopL with Alkaline Phosphatases (APases)-Tobacco leaves were infiltrated with A. tumefaciens AGL1 carrying pPZP-nopL (24). Three days later, proteins were extracted from tobacco leaf discs as previously described (23) without EDTA, using 10 mM MgCl 2 instead of KCl.…”

“…Necrotic areas and infiltrated areas of tobacco leaves were quantified with the Adobe Acrobat 8 program. Where indicated, transformation of tobacco leaves was performed with strain AGL1 harboring plasmid pPZP-nopL (24), which resulted in nopL expression driven by the CaMV 35 S-promotor. Bioinformatic Analysis-Putative phosphorylation sites of nopL were predicted with the NetPhos 2.0 Server, and kinasespecific phosphorylation sites of nopL with the NetPhosK 1.0 Server from the Technical University of Denmark.…”

Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

“…Meanwhile, the T3Es NopL and NopP of NGR234 have positive effects on symbiosis with their host legumes (9). Both NopL and NopP are phosphorylated by plant protein kinases, and NopL can prevent the complete induction of pathogenesis-related defense proteins when expressed in Lotus japonicus roots (10). However, the molecular basis for T3SS-involved nodulation enhancement has not been fully elucidated.…”