Genomic dissection of ROS detoxifying enzyme encoding genes for their role in antioxidative defense mechanism against Tomato leaf curl New Delhi virus infection in tomato

In the present study, genes encoding for six major classes of enzymatic antioxidants, namely superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), Peroxidase (Prx) and glutathione S-transferase (GST) are identified in tomato. Their expression was studied in tomato cultivars contrastingly tolerant to ToLCNDV during virus infection and different hormone treatments. Significant upregulation of SlGR3, SlPrx25, SlPrx75, SlPrx95, SlGST44, and SlGST96 was observed in the tolerant cultivar during disease infection. Virus-induced gene silencing of SlGR3 in the tolerant cultivar conferred disease susceptibility to the knock-down line, and higher accumulation (~80%) of viral DNA was observed in the tolerant cultivar. Further, subcellular localization of SlGR3 showed its presence in cytoplasm, and its enzymatic activity was found to be increased (~65%) during ToLCNDV infection. Knock-down lines showed ~3- and 3.5-fold reduction in GR activity, which altogether underlines that SlGR3 is vital component of the defense mechanism against ToLCNDV infection.     

Quantitative proteomic analyses reveal that RBBI3.3, a trypsin inhibitor protein,plays an important role in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> infection in rice

Magnaporthe oryzae (M. oryzae) is the causative agent of rice blast, the most destructive rice disease in China. This study was designed to ascertain the molecular mechanisms of the response of rice to M. oryzae infection to facilitate the breeding of new high-quality and disease-resistant rice varieties using isobaric tags for relative and absolute quantification (iTRAQ) combined with a high-throughput mass spectrometry identification platform. M. oryzae infection models were constructed with the resistant rice cultivar Gumei2 and the non-resistant cultivar Lijiangxintuanheigu (LTH). The results showed that total of 1541 proteins were identified, among which 843 proteins were overlapping between the two biological replicates analyses. Seventy-one proteins were classified as fungi-responsive. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed the important roles of these proteins in metabolic processes. Detailed phenotypic analyses revealed that the trypsin inhibitor RBBI3.3 was effective in inhibiting the initial formation of appressoria. Our quantitative proteomic study provides insights into the molecular mechanism underlying M. oryzae resistance in the incompatible rice Gumei2. The identification of RBBI3.3 as a key defense regulator highlights a new possibility for disease control.     

<Emphasis Type="Italic">Wsi18</Emphasis> promoter from wild rice genotype, <Emphasis Type="Italic">Oryza nivara</Emphasis>, shows enhanced expression under soil water stress in contrast to elite cultivar, <Emphasis Type="Italic">IR20</Emphasis>

Identification and characterization of plant promoters from wild rice genotypes showing inducible expression under soil water stress (SWS) is desirable for transgene expression to generate stress tolerant rice cultivars. A comparative expression profiling of Wsi18, a group 3 LEA gene, revealed differential response under SWS conditions between modern cultivated rice (IR20) and its wild progenitor (Oryza nivara). Wsi18 promoter from O. nivara showed enhanced inducible expression of the reporter gusA gene, encoding β-glucuronidase, in transgenic rice plants in comparison to similar promoter from IR20. Deletion analysis unravelled the cis-acting regulatory elements minimally required for optimal expression of Wsi18 promoter from O. nivara under SWS condition. This is the first report of characterization of an inducible promoter from a wild rice genotype to drive the gene expression under water stress conditions. The Wsi18 promoter element from the wild rice genotype can be used in future genetic manipulation strategies for the generation of SWS tolerant rice cultivars with improved yield characteristics.     

Improved tolerance against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> in transgenic tomato over-expressing multi-domain proteinase inhibitor gene from <Emphasis Type="Italic">Capsicum annuum</Emphasis>

Plant proteinase inhibitors (PIs) are plant defense proteins and considered as potential candidates for engineering plant resistances against herbivores. Capsicum annuum proteinase inhibitor (CanPI7) is a multi-domain potato type II inhibitor (Pin-II) containing four inhibitory repeat domains (IRD), which target major classes of digestive enzymes in the gut of Helicoverpa armigera larvae. Stable integration and expression of the transgene in T1 transgenic generation, were confirmed by established molecular techniques. Protein extract of transgenic tomato lines showed increased inhibitory activity against H. armigera gut proteinases, supporting those domains of CanPI7 protein to be effective and active. When T1 generation plants were analyzed, they exhibited antibiosis effect against first instar larvae of H. armigera. Further, larvae fed on transgenic tomato leaves showed delayed growth relative to larvae fed on control plants, but did not change mortality rates significantly. Thus, better crop protection can be achieved in transgenic tomato by overexpression of multi-domain proteinase inhibitor CanPI7 gene against H. armigera larvae.     

SlAGO4A,a core factor of RNA-directed DNA methylation (RdDM) pathway,plays an important role under salt and drought stress in tomato

In Arabidopsis, it has been clarified that AGO4 protein is implicated in a phenomenon termed RNA-directed DNA methylation (RdDM). Previously, four orthologs of AtAGO4 were cloned in tomato, designated as SlAGO4A–SlAGO4D. Here, we studied the role of the SlAGO4A gene in regulating salt and drought tolerance in tomato. SlAGO4A-down-regulating (AS) transgenic tomato plants showed enhanced tolerance to salt and drought stress compared to wild-type (WT) and SlAGO4A-overexpressing (OE) transgenic plants, as assessed by physiological parameters such as seed germination rate, primary root length, chlorophyll/proline/MDA/soluble sugar/RWC content, and survival rate. Moreover, several genes involved in ROS scavenging and plant defense, including CAT, SOD, GST, POD, APX, LOX, and PR1, were up- or down-regulated consistently under salt and drought stress. Notably, expression levels of some DNA methyltransferase genes and RNAi pathway genes were significantly lower in AS plants than in WT. Taken together, our results suggest that SlAGO4A gene plays a negative role under salt and drought stress in tomato probably through the modulation of DNA methylation as well as the classical RNAi pathway. Hence, it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.     

Root nematode infection enhances leaf defense against whitefly in tomato

The foliar response to different herbivores sharing the same hosts is an important topic for the study of plant-insect interactions. Plants evolve local and systemic resistant strategies to cope with herbivores. Many researchers have characterized the mechanisms of leaf responses to insect infestation; however, the fact that roots serve as systemic resistance modulators to leaf herbivores has been widely ignored. Here, we report that tomato (Solanum lycopersicum) plants infected with southern root-knot nematodes (Meloidogyne incognita)—which feed on the roots to form nodules—enhanced leaf defenses against aboveground attackers, specifically, the whitefly (Bemisia tabaci). Our results show that nematode infection reduced the whitefly population abundance because of conferring a stronger SA-dependent defense pathway against whitefly than in tomato plants without nematode infection. Meanwhile, nematode-infected tomato plant also activated the foliar JA-dependent defense pathway at 4 h after whitefly infestation. However, the foliar JA-dependent defense under whitefly infestation alone was suppressed, with the JA content being nearly 30 % lower than that in tomato plants co-infected with nematodes and whiteflies. Furthermore, nematode infection significantly decreased the plant nitrogen concentration in leaves and roots. As a result, nematode infection reduced the number of whiteflies by enhancing foliar SA-dependent defense, activating JA-dependent defense and decreasing nitrogen nutrition. Our results suggest that underground nematode infection significantly enhances the defense ability of tomato plants against whitefly.     

Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

Identification of genes differentially expressed in husk tomato (<Emphasis Type="Italic">Physalis philadelphica</Emphasis>) in response to whitefly (<Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis>) infestation

Plants respond to phloem-feeding whiteflies by extensive changes in gene expression. To identify differentially expressed genes in husk tomato plants (Physalis philadelphica) infested with Trialeurodes vaporariorum, young plants were challenged with adult whiteflies, and forward and reverse subtractive libraries were constructed from infested leaves at 5 and 15 days after infestation. Several genes were identified as up-regulated; these included a diversity of genes involved in plant defense responses, protein synthesis or degradation, and cell wall fortification or modification. Genes required for amino acid biosynthesis, lipid metabolism and synthesis, including cell surface components such as suberin, responses to stress, photosynthesis and other functions, were similarly induced. Down-regulated genes were also identified, most prominently kinases and aquaporin genes. Similarities in defense responses between tomato and P. philadelphica were noted regarding the expression of certain genes in response to nematode, aphid, or whitefly. A role for abscisic acid, brassinosteroids, and cytokinins in the regulated response to whitefly infestation in P. philadelphica was also implied by the expression pattern of phytohormone-associated genes, including genes coding for proteins containing F-box motifs. Differential expression of selected genes was validated by quantitative real-time PCR. The possible role played by some of these genes during whitefly infestation is discussed.     

Identification of a molecular marker tightly linked to bacterial wilt resistance in tomato by genome-wide SNP analysis

Key message

Genotyping of disease resistance to bacterial wilt in tomato by a genome-wide SNP analysis

Abstract

Bacterial wilt caused by Ralstonia pseudosolanacearum is one of the destructive diseases in tomato. The previous studies have identified Bwr-6 (chromosome 6) and Bwr-12 (chromosome 12) loci as the major quantitative trait loci (QTLs) contributing to resistance against bacterial wilt in tomato cultivar ‘Hawaii7996’. However, the genetic identities of two QTLs have not been uncovered yet. In this study, using whole-genome resequencing, we analyzed genome-wide single-nucleotide polymorphisms (SNPs) that can distinguish a resistant group, including seven tomato varieties resistant to bacterial wilt, from a susceptible group, including two susceptible to the same disease. In total, 5259 non-synonymous SNPs were found between the two groups. Among them, only 265 SNPs were located in the coding DNA sequences, and the majority of these SNPs were located on chromosomes 6 and 12. The genes that both carry SNP(s) and are near Bwr-6 and Bwr-12 were selected. In particular, four genes in chromosome 12 encode putative leucine-rich repeat (LRR) receptor-like proteins. SNPs within these four genes were used to develop SNP markers, and each SNP marker was validated by a high-resolution melting method. Consequently, one SNP marker, including a functional SNP in a gene, Solyc12g009690.1, could efficiently distinguish tomato varieties resistant to bacterial wilt from susceptible varieties. These results indicate that Solyc12g009690.1, the gene encoding a putative LRR receptor-like protein, might be tightly linked to Bwr-12, and the SNP marker developed in this study will be useful for selection of tomato cultivars resistant to bacterial wilt.

     

Contribution of the non-effector members of the HrpL regulon, <Emphasis Type="Italic">iaaL</Emphasis> and <Emphasis Type="Italic">matE</Emphasis>, to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. tomato DC3000 in tomato plants

Background

The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.

Results

A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.

Conclusions

Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

     

Comparative proteomic analysis of leaf tissue from tomato plants colonized with <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis>

A comparative proteomic approach was performed to analyze the differential accumulation of leaf proteins in response to the symbiosis between Solanum lycopersicum and the arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis. Protein profiling was examined in leaves from tomato plants colonized with AMF (M), as well as non-colonized plants fertilized with low phosphate (20 μM P; NM-LP) and non-colonized plants fertilized with regular phosphate Hoagland’s solution (200 μM P; NM-RP). Comparisons were made between these groups, and 2D-SDS-PAGE revealed that 27 spots were differentially accumulated in M vs. NM-LP. Twenty-three out of the 27 spots were successfully identified by mass spectrometry. Two of these proteins, 2-methylene-furan-3-one reductase and auxin-binding protein ABP19a, were up-accumulated in M plants. The down-accumulated proteins in M plants were associated mainly with photosynthesis, redox, and other molecular functions. Superoxide dismutase, harpin binding protein, and thioredoxin peroxidase were down-accumulated in leaves of M tomato plants when compared to NM-LP and NM-RP, indicating that these proteins are responsive to AMF colonization independently of the phosphate regime under which they were grown. 14-3-3 protein was up-accumulated in NM-RP vs. NM-LP plants, whereas it was down-accumulated in M vs. NM-LP and M vs. NM-RP, regardless of their phosphate nutrition. This suggests a possible regulation by P nutrition and AMF colonization. Our results demonstrate AMF-induced systemic changes in the expression of tomato leaf proteins, including the down-accumulation of proteins related to photosynthesis and redox function.     

Characterization and evaluation of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain WF02 regarding its biocontrol activities and genetic responses against bacterial wilt in two different resistant tomato cultivars

Bacillus amyloliquefaciens strain WF02, isolated from soil collected at Wufeng Mountain, Taiwan, has siderophore-producing ability and in vitro antagonistic activity against bacterial wilt pathogen. To determine the impact of plant genotype on biocontrol effectiveness, we treated soil with this strain before infecting susceptible (L390) and moderately resistant (Micro-Tom) tomato cultivars with Ralstonia solanacearum strain Pss4. We also compared the efficacy of this strain with that of commercial Bacillus subtilis strain Y1336. Strain WF02 provided longer lasting protection against R. solanacearum than did strain Y1336 and controlled the development of wilt in both cultivars. To elucidate the genetic responses in these plants under WF02 treatment, we analyzed the temporal expression of defense-related genes in leaves. The salicylic acid pathway-related genes phenylalanine ammonia-lyase and pathogenesis-related protein 1a were up-regulated in both cultivars, whereas expression of the jasmonic acid pathway-related gene lipoxygenase was only elevated in the susceptible tomato cultivar (L390). These results suggest that WF02 can provide protection against bacterial wilt in tomato cultivars with different levels of disease resistance via direct and indirect modes of action.