A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.     

Avr/Cf互作诱发番茄Pto互作因子Pti编码基因的表达

植物抗病基因Pto和Cf产物具完全不同的结构域,其细胞定位也不同.二者决定的抗病性产生机制的异同令人关注.采用两种过敏性反应(hypersensitive response,HR)产生系统研究了Pto互作蛋白Pti4、Pti5和Pti6编码基因在Avr/Cf互作中的时序表达:(1)通过杂交方法获取同含互补基因对Avr4/Cf-4和Avr9/Cf-9的番茄(Lycopersicon esculentum Mill.)种子.常温下这些种子发芽后形成的苗产生HR坏死斑.(2)先将Avr/Cf苗置于33 ℃下培养,此时番茄苗生长正常,不形成HR坏死斑.然后将温度降至25 ℃,数小时内这些苗即形成HR坏死斑.不同方法研究结果均表明,随Avr/Cf苗中HR坏死斑的形成,Pti4、Pti5和Pti6均受显著诱导表达.但它们的表达水平和动态不同.这些结果表明,这些Pti在功能上互补,可能同时涉及Pto和Cf决定性抗性的调节作用.     

Pto mutants differentially activate Prf-dependent,avrPto-independent resistance and gene-for-gene resistance

Pto confers disease resistance to Pseudomonas syringae pv tomato carrying the cognate avrPto gene. Overexpression of Pto under the cauliflower mosaic virus 35S promoter activates spontaneous lesions and confers disease resistance in tomato (Lycopersicon esculentum) plants in the absence of avrPto. Here, we show that these AvrPto-independent defenses require a functional Prf gene. Several Pto-interacting (Pti) proteins are thought to play a role in Pto-mediated defense pathways. To test if interactions with Pti proteins are required for the AvrPto-independent defense responses by Pto overexpression, we isolated several Pto mutants that were unable to interact with one or more Pti proteins, but retained normal interaction with AvrPto. Overexpression of two mutants, Pto(G50S) and Pto(R150S), failed to activate AvrPto-independent defense responses or confer enhanced resistance to the virulent P. s. pv tomato. When introduced into plants carrying 35S::Pto, 35S::Pto(G50S) dominantly suppressed the AvrPto-independent resistance caused by former transgene. 35S::Pto(G50S) also blocked the induction of a number of defense genes by the wild-type 35S::Pto. However, 35S::Pto(G50S) and 35S::Pto(R150S) plants were completely resistant to P. s. pv tomato (avrPto), indicating a normal gene-for-gene resistance. Furthermore, 35S::Pto(G50S) plants exhibited normal induction of defense genes in recognition of avrPto. Thus, the AvrPto-independent defense activation and gene-for-gene resistance mediated by Pto are functionally separable.     

Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

The Pto gene was derived originally from the wild tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pto and fen. High conservation of genome organization between the two tomato species allowed us to identify the pto and fen alleles from among the cluster of closely related Pto gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Pti1 serine/threonine kinase. However, the pto kinase shows impaired interaction with Pti1 and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto- and Fen-mediated signal transduction pathways.     

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.     

Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

The PTI1-like kinase ZmPti1a from maize (Zea mays L.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte

Background  

The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR) reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied.     

Two expressed soybean genes with high sequence identity to tomato Pti1 kinase lack autophosphorylation activity

An important signaling pathway for disease resistance in tomato involves the R gene product Pto which phosphorylates Ptil, a downstream member of this signaling cascade. Both Pto and Pti1 are Ser/Thr protein kinases capable of autophosphorylation in vitro. Two soybean (Glycine max L. Merr. var. Hobbit) cDNAs (sPti1a and sPti1b) were cloned and sequenced and found to each have 78% amino acid sequence identity with tomato Pti1. Glutathione S-transferase fusions of sPti1a and b expressed in Escherichia coli did not autophosphorylate in vitro, but were efficiently phosphorylated by tomato Pto. Replacement of Tyr197 with an Asp that is invariant at this position in other protein kinases did not restore autophosphorylation activity to sPti1a or b. Tyr197 was also present in the Pti1 homologues of three distant relatives of G. max. Together these results suggest that soybean Pti1 might function in a Pto-like signaling pathway that does not require Pti1 kinase activity.     

The major site of the pti1 kinase phosphorylated by the pto kinase is located in the activation domain and is required for pto-pti1 physical interaction.

The Pto and Pti1 serine/threonine protein kinases are key components of the signaling pathway leading to speck disease resistance in tomato. The two kinases physically interact in the yeast two-hybrid system, and Pto specifically phosphorylates Pti1 in vitro. In this study, we identified and characterized the major Pti1 site phosphorylated by Pto. Pto was expressed in Escherichia coli as a maltose-binding fusion protein (MBP-Pto), and used to phosphorylate in vitro a kinase deficient Pti1 protein fused to glutathione S-transferase (GST-Pti1[K96N]). The major phosphopeptide derived from trypsin digestion of phosphorylated GST-Pti1(K96N) was partially purified by reverse-phase HPLC and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Its mass corresponded to phosphopeptide LHSTR, which lies in the Pti1 kinase activation domain at amino acid position 230-234. By phosphoamino acid analysis, Thr233 was determined to be the phosphorylation site of peptide LHSTR. Mutations of Thr233 reduced dramatically Pti1 phosphorylation by MBP-Pto and Pti1 autophosphorylation, providing evidence that the same Pti1 site is involved in the two reactions. Moreover, phosphorylation of Thr233 appeared to be required for Pto-Pti1 physical interaction, as a mutation of this site to alanine, but not to aspartate, abolished the interaction between Pto and Pti1 in the yeast two-hybrid system.     

Molecular mechanisms involved in bacterial speck disease resistance of tomato

An important recent advance in the field of plant-microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or nematodes. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. The future challenge is to understand how R gene products specifically perceive defence-eliciting signals from the pathogen and transduce those signals to pathways that lead to the activation of plant defence responses. In tomatoes, the Pto kinase (product of the Pto R gene) confers resistance to strains of the bacterial speck pathogen, Pseudomonas syringae pv. tomato, that carry the corresponding avirulence gene avrPto. Resistance to bacterial speck disease is initiated by a mechanism involving the physical interaction of the Pto kinase and the AvrPto protein. This recognition event initiates signalling events that lead to defence responses including an oxidative burst, the hypersensitive response and expression of pathogenesis-related genes. Pto-interacting (Pti) proteins have been identified that appear to act downstream of the Pto kinase and our current studies are directed at elucidating the roles of these components.     

Plant Defense Genes Are Synergistically Induced by Ethylene and Methyl Jasmonate

Combinations of ethylene and methyl jasmonate (E/MeJA) synergistically induced members of both groups 1 and 5 of the pathogenesis-related (PR) superfamily of defense genes. E/MeJA caused a synergistic induction of PR-1b and osmotin (PR-5) mRNA accumulation in tobacco seedlings. E/MeJA also synergistically activated the osmotin promoter fused to a [beta]-glucuronidase marker gene in a tissue-specific manner. The E/MeJA responsiveness of the osmotin promoter was localized on a -248 to +45 fragment that exhibited responsiveness to several other inducers. E/MeJA induction also resulted in osmotin protein accumulation to levels similar to those induced by osmotic stress. Of the several known inducers of the osmotin gene, including salicylic acid (SA), fungal infection is the only other condition known to cause substantial osmotin protein accumulation in Wisconsin 38, a tobacco cultivar that does not respond hypersensitively to tobacco mosaic virus. Based on the ability of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine to block ethylene induction of PR-1b mRNA accumulation and its inability to block osmotin mRNA induction by ethylene, these two PR gene groups appeared to have at least partially separate signal transduction pathways. Stimulation of osmotin mRNA accumulation by okadaic acid indicated that another protein kinase system is involved in regulation of the osmotin gene. SA, which is known to induce pathogen resistance in tobacco, could not induce the osmotin gene as much as E/MeJA and neither could it induce PR-1b as much as SA and MeJA combined.