Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici

MicroRNAs (miRNAs) play very important roles in plant defense responses. However, little is known about their roles in the susceptibility interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). In this study, two miRNA libraries were constructed from the leaves of the cultivar Xingzi 9104 inoculated with the virulent Pst race CYR32 and sterile water, respectively. A total of 1316 miRNA candidates, including 173 known miRNAs that were generated from 98 pre‐miRNAs, were obtained. The remaining 1143 miRNA candidates included 145 conserved and 998 wheat‐specific miRNAs that were generated from 87 and 1088 pre‐miRNAs, respectively. The 173 known and 145 conserved miRNAs were sub‐classified into 63 miRNA families. The target genes of wheat miRNAs were also confirmed using degradome sequencing technology. Most of the annotated target genes were related to signal transduction or energy metabolism. Additionally, we found that miRNAs and their target genes form complicated regulation networks. The expression profiles of miRNAs and their corresponding target genes were further analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the results indicate that some miRNAs are involved in the compatible wheat‐Pst susceptibility interaction. Importantly, tae‐miR1432 was highly expressed when wheat was challenged with CYR32, and the corresponding target gene, predicted to be a calcium ion‐binding protein, also exhibited upregulated expression but a divergent expression trend. PC‐3P‐7484, a specific wheat miRNA, was highly expressed in the wheat response to Pst infection, while the expression of the corresponding target gene ubiquillin was dramatically downregulated. These data provide the foundation for evaluating the important regulatory roles of miRNAs in wheat‐Pst susceptibility interaction.     

Differential expression of genes encoding calmodulin-binding proteins in response to bacterial pathogens and inducers of defense responses

Calmodulin (CaM) plays an important role in sensing and transducing changes in cellular Ca²⁺ concentration in response to several biotic and abiotic stresses. Although CaM is implicated in plant-pathogen interactions, its molecular targets and their role in defense signaling pathway(s) are poorly understood. To elucidate the signaling pathways that link CaM to defense responses, we screened a cDNA library constructed from bean leaves undergoing a hypersensitive response (HR) with radiolabeled CaM isoforms. A total of 26 putative CBPs were identified. Sequencing of the cDNAs revealed that they represent 8 different genes. They are homologues of previously identified CaM-binding proteins (CBPs) in other systems. However, some CBPs are novel members of known CBP families. The proteins encoded by these clones bound CaM in a Ca²⁺-dependent manner. To determine if these CBPs are involved in plant defense responses, we analyzed their expression in bean leaves inoculated with compatible, incompatible and nonpathogenic bacterial strains. Expression of three CBPs including an isoform of cyclic nucleotide-gated channels (PvCNGC-A) and two hypothetical proteins (PvCBP60-C and PvCBP60-D) was induced whereas the expression of two other isoforms of CNGCs (PvCNGC-Band PvCNGC-C) was repressed in response to incompatible pathogens. The expression of the rest, a small auxin up RNA (PvSAUR1) and two hypothetical proteins (PvCBP60-Aand PvCBP60-B), was not changed. The expression of most of the pathogen-regulated genes was also affected by salicylic acid, jasmonic acid, hydrogen peroxide and a fungal elicitor, which are known to induce defense responses. Our results strongly suggest that at least five bean CBPs are involved in plant defense responses.     

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.     

The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley

Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be expressed in pathogen-attacked epidermal tissue of barley and wheat leaves, and the corresponding proteins are proposed to accumulate in the apoplast. Here, the role of HvGLP4 and TaGLP4 in the defense of barley and wheat against Blumeria graminis (DC.) E. O. Speer, the cereal powdery mildew fungus, was examined in an epidermal transient expression system and in transgenic Arabidopsis thaliana plants overexpressing His-tagged HvGLP4. Leaf extracts of transgenic Arabidopsis overexpressing HvGLP4 contained a novel His-tagged protein with superoxide dismutase activity and HvGLP4 epitopes. Transient overexpression of TaGLP4 and HvGLP4 enhanced resistance against B. graminis in wheat and barley, whereas transient silencing by RNA interference reduced basal resistance in both cereals. The effect of GLP4 overexpression or silencing was strongly influenced by the genotype of the plant. The data suggest that members of GLP subfamily 4 are components of quantitative resistance in both barley and wheat, acting together with other, as yet unknown, plant components.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya.

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.     

Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in activation of plant defense responses and localized cell death

The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades.     

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.     

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.