Purification and phosphorylation of the effector protein NopL from Rhizobium sp. NGR234

Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells. These effectors perturb host-defence responses, especially signal transduction pathways. A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp. NGR234. NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes. To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants. NopL serves as a substrate for plant protein kinases as well as purified protein kinase A. Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.     

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

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 NGRΩnopL (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 (α-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.     

Characterization of Nops,nodulation outer proteins,secreted via the type III secretion system of NGR234

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.     

NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.     

Removal of Vacuolar Targeting Signal from Class I Vacuolar Chitinase leads to its Extracellular Secretion in Transgenic Tobacco

Chitinase, an antifungal pathogenesis related (PR) protein is present in different isoforms. Class I basic chitinase which is generally more antifungal in nature compared to other chitinase classes, is present in vacuoles. It is speculated that extracellular secretion of this vacuolar enzyme by removing its vacuolar targeting signal at C- terminus might further increase its effectivity. Tobacco class I chitinase cDNA was earlier modified by PCR to add two stop codons before vacuolar targeting signal, so that the protein without this signal would be secreted extracellularly.Transgenic tobacco plants were raised with modified chitinase cDNA and native unmodified cDNA, both under the control of CaMV 35 S promoter, using Agrobacterium mediated transformation. Transgenic plants with unmodified class I chitinase cDNA expressed the enzyme in vacuoles and those having modified cDNA expressed the enzyme in extracellular spaces while retaining its biological activity.     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.     

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.     

Wound-induced expression of a tobacco peroxidase is not enhanced by ethephon and suppressed by methyl jasmonate and coronatine

In tobacco plants, wounding induces production of a set of defense-related proteins such as basic pathogenesis-related (PR) proteins and proteinase inhibitors (PIs) via the jasmonate/ethylene pathway. Although class III plant peroxidase (POX) is also wound-inducible, the regulatory mechanism for its wound-induced expression is not fully understood. Here, we describe that a tobacco POX gene (tpoxN1), which is constitutively expressed in roots, is induced locally 30 min after wounding and then systemically in tobacco plants. Infection of necrotizing virus also induced tpoxN1 gene. The wound-induced expression was not enhanced by known wound-signal compounds such as methyl jasmonate (MeJA) and ethephon in contrast to other wound-inducible genes such as basic PR-1 and PI-II genes. And treatment with MeJA and coronatine, biological analogs of jasmonate, rather suppressed the tpoxN1 expression. Salicylic acid, an antagonist of jasmonate-based wound signaling, did not suppress the wound-induced expression of tpoxN1. Only spermine, which is reported as an endogenous inducer for acidic PR genes in tobacco mosaic virus-infected tobacco leaves, could induce tpoxN1 gene expression. These results suggest that wound-induced expression of the tpoxN1 gene is regulated differently from that of the basic PR and PI-II genes.     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast

The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.     

A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter

Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.     

小麦几丁质酶基因的异种表达及其功能鉴定

几丁质酶参与植物的发育及防卫反应,并与人类疾病发生有关.文章研究了小麦几丁质酶基因Wch2经根癌农杆菌介导的烟草瞬间表达和转基因拟南芥的稳定表达,Western杂交及酶活测定证实,瞬间表达的小麦几丁质酶分子量约30 kD,具有降解几丁质多聚物的功能;Wch2在转入拟南芥后表达量高,尖孢镰刀菌接种的鉴定表明,表达Wch2的转基因植株的抗病性显著高于表达绿色荧光蛋白的对照植株.这些结果说明Wch2的异种表达,可用于植物抗病基因工程,以增强植物的抗病性.