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Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling.

ABSTRACT: Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by strain NGR?nopL (NGR234 mutated in nopL), indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of nopL in yeast disrupted the mating pheromone (?-factor) response pathway, whereas nopL expression in N. tabacum suppressed cell death induced either by overexpression of the MAPK gene SIPK (salicylic acid-induced protein kinase) or by SIPK(DD) (mutation in the TXY motif resulting in constitutive MAPK activity). These data indicate that NopL impaired function of MAPK proteins or MAPK substrates. Furthermore, we demonstrate that NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro pattern, a typical motif in MAPK substrates. Taken together, data suggest that NopL mimics a MAPK substrate and that NopL suppresses premature nodule senescence by impairing MAPK signaling in host cells.

SUBMITTER: Zhang L

PROVIDER: S-EPMC3173237 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling.

Zhang Ling L Chen Xue-Jiao XJ Lu Huang-Bin HB Xie Zhi-Ping ZP Staehelin Christian C

The Journal of biological chemistry 20110720 37

Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodule ...[more]

PMID: 21775427

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Project description:Rhizobia can infect legumes and induce the coordinated expression of symbiosis and defense genes for the establishment of mutualistic symbiosis. Numerous studies have elucidated the molecular interactions between rhizobia and host plants, which are associated with Nod factor, exopolysaccharide, and T3SS effector proteins. However, there have been relatively few reports about how the host plant recognizes the outer membrane proteins (OMPs) of rhizobia to mediate symbiotic nodulation. In our previous work, a gene (Mhopa22) encoding an OMP was identified in Mesorhizobium huakuii 7653R, whose homologous genes are widely distributed in Rhizobiales. In this study, a germin-like protein GLP1 interacting with Mhopa22 was identified in Astragalus sinicus. RNA interference of AsGLP1 resulted in a decrease in nodule number, whereas overexpression of AsGLP1 increased the number of nodules in the hairy roots of A. sinicus. Consistent symbiotic phenotypes were identified in Medicago truncatula with MtGLPx (refer to medtr7g111240.1, the isogeny of AsGLP1) overexpression or Tnt1 mutant (glpx-1) in symbiosis with Sinorhizobium meliloti 1021. The glpx-1 mutant displayed hyperinfection and the formation of more infection threads but a decrease in root nodules. RNA sequencing analysis showed that many differentially expressed genes were involved in hormone signaling and symbiosis. Taken together, AsGLP1 and its homology play an essential role in mediating the early symbiotic process through interacting with the OMPs of rhizobia. IMPORTANCE This study is the first report to characterize a legume host plant protein to sense and interact with an outer membrane protein (OMP) of rhizobia. It can be speculated that GLP1 plays an essential role to mediate early symbiotic process through interacting with OMPs of rhizobia. The results provide deeper understanding and novel insights into the molecular interactive mechanism of a legume symbiosis signaling pathway in recognition with rhizobial OMPs. Our findings may also provide a new perspective to improve the symbiotic compatibility and nodulation of legume.

Project description:BACKGROUND: In nitrate-poor soils, many leguminous plants form nitrogen-fixing symbioses with members of the bacterial family Rhizobiaceae. We selected Rhizobium sp. NGR234 for its exceptionally broad host range, which includes more than I 12 genera of legumes. Unlike the genome of Bradyrhizobium japonicum, which is composed of a single 8.7 Mb chromosome, that of NGR234 is partitioned into three replicons: a chromosome of about 3.5 Mb, a megaplasmid of more than 2 Mb (pNGR234b) and pNGR234a, a 536,165 bp plasmid that carries most of the genes required for symbioses with legumes. Symbiotic loci represent only a small portion of all the genes coded by rhizobial genomes, however. To rapidly characterize the two largest replicons of NGR234, the genome of strain ANU265 (a derivative strain cured of pNGR234a) was analyzed by shotgun sequencing. RESULTS: Homology searches of public databases with 2,275 random sequences of strain ANU265 resulted in the identification of 1,130 putative protein-coding sequences, of which 922 (41%) could be classified into functional groups. In contrast to the 18% of insertion-like sequences (ISs) found on the symbiotic plasmid pNGR234a, only 2.2% of the shotgun sequences represent known ISs, suggesting that pNGR234a is enriched in such elements. Hybridization data also indicate that the density of known transposable elements is higher in pNGR234b (the megaplasmid) than on the chromosome. Rhizobium-specific intergenic mosaic elements (RIMEs) were found in 35 shotgun sequences, 6 of which carry RIME2 repeats previously thought to be present only in Rhizobium meliloti. As non-overlapping shotgun sequences together represent approximately 10% of ANU265 genome, the chromosome and megaplasmid may carry a total of over 200 RIMEs. CONCLUSIONS: 'Skimming' the genome of Rhizobium sp. NGR234 sheds new light on the fine structure and evolution of its replicons, as well as on the integration of symbiotic functions in the genome of a soil bacterium. Although most putative coding sequences could be distributed into functional classes similar to those in Bacillus subtilis, functions related to transposable elements were more abundant in NGR234. In contrast to ISs that accumulated in pNGR234a and pNGR234b, the hundreds of RIME elements seem mostly attributes of the chromosome.

Dataset Information

Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling.

Publications

Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling.

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