Project description:Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP) techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers, sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular events underlying the HSR of maturing microspores of a crop plant, tomato.
Project description:Salmonella enterica can colonize all parts of the tomato plant. Tomatoes have been frequently implicated in salmonellosis outbreaks. In agricultural settings, Salmonella must overcome stress, nutritional and competition barriers to become established on plant surfaces. Knowledge of the genetic mechanisms underlying Salmonella-plant associations is limited, especially when growing epiphytically. A genome-wide transcriptomic analysis of Salmonella Typhimurium (SeT) was conducted with RNA-Seq to elucidate strategies for epiphytic growth on live, intact tomato shoot and root surfaces. Six plasmid-encoded and 123 chromosomal genes were significantly (using Benjamini-Hochberg adjusted p-values) up-regulated; 54 and 110 detected in SeT on shoots and roots, respectively, with 35 common to both. Key signals included NsrR regulon genes needed to mitigate nitrosative stress, oxidative stress genes and host adaptation genes, including environmental stress, heat shock and acid-inducible genes. Several amino acid biosynthesis genes and genes indicative of sulphur metabolism and anaerobic respiration were up-regulated. Some Type III secretion system (T3SS) effector protein genes and their chaperones from pathogenicity island-2 were expressed mostly in SeT on roots. Gene expression in SeT was validated against SeT and also the tomato outbreak strain Salmonella Newport with a high correlation (R 2 = 0.813 and 0.874, respectively; both p < 0.001). Oxidative and nitrosative stress response genes, T3SS2 genes and amino acid biosynthesis may be needed for Salmonella to successfully colonize tomato shoot and root surfaces.
Project description:The footprint of tomato cultivation, a cool region crop that exhibits heat stress (HS) sensitivity, is increasing in the tropics/sub-tropics. Knowledge of novel regulatory hot spots from varieties growing in the Indian sub-continent climatic zones could be vital for developing HS-resilient crops. Comparative transcriptome-wide signatures of a tolerant (CLN1621L) and sensitive (CA4) cultivar pair shortlisted from a pool of varieties exhibiting variable thermo-sensitivity using physiological-, survival- and yield-related traits revealed redundant to cultivar-specific HS regulation. The antagonistically expressing genes encode enzymes and proteins that have roles in plant defence and abiotic stresses. Functional characterization of three antagonistic genes by overexpression and silencing established Solyc09g014280 (Acylsugar acyltransferase) and Solyc07g056570 (Notabilis) that are up-regulated in tolerant cultivar, as positive regulators of HS tolerance and Solyc03g020030 (Pin-II proteinase inhibitor), that are down-regulated in CLN1621L, as negative regulator of thermotolerance. Transcriptional assessment of promoters of these genes by SNPs in stress-responsive cis-elements and promoter swapping experiments in opposite cultivar background showed inherent cultivar-specific orchestration of transcription factors in regulating transcription. Moreover, overexpression of three ethylene response transcription factors (ERF.C1/F4/F5) also improved HS tolerance in tomato. This study identifies several novel HS tolerance genes and provides proof of their utility in tomato thermotolerance.
Project description:Gene expression cascade in a plant is altered in times of stress. Reprogramming of the expression profiles of genes is required for a robust and specific response. Until now, tomato transcriptomic alteration in response to Alternaria fungal stress were not known. This study presents the profile of genes that are differentially expressed during Alternaria stress in local tomato cultivar (Pusa Ruby). At least 2944 genes expression was varied and pathways that are altered during this compatible interaction have been identified. Supported by DBT, Govt. of India. Overall design: RNA seq profile of Indian tomato (Solanum lycopersicum) cultivar Pusa Ruby infected with Alternaria. One mock infected and One Alternaria fungus infected samples were analyzed.
Project description:The present study investigated the transcriptomic and metabolomic changes elicited in tomato plants (Solanum lycopersicum cv. Micro-Tom) following treatments with the biocontrol agent Trichoderma harzianum strain M10 or its purified secondary metabolite harzianic acid (HA), in the presence or the absence of the soil-borne pathogen Rhizoctonia solani. Transcriptomic analysis allowed the identification of differentially expressed genes (DEGs) that play a pivotal role in resistance to biotic stress. Overall, the results support the ability of T. harzianum M10 to activate defense responses in infected tomato plants. An induction of hormone-mediated signaling was observed, as shown by the up-regulation of genes involved in the ethylene and jasmonate (ET/JA) and salicylic acid (SA)-mediated signaling pathways. Further, the protective action of T. harzianum on the host was revealed by the over-expression of genes able to detoxify cells from reactive oxygen species (ROS). On the other hand, HA treatment also stimulated tomato response to the pathogen by inducing the expression of several genes involved in defense response (including protease inhibitors, resistance proteins like CC-NBS-LRR) and hormone interplay. The accumulation of steroidal glycoalkaloids in the plant after treatments with either T. harzianum or HA, as determined by metabolomic analysis, confirmed the complexity of the plant response to beneficial microbes, demonstrating that these microorganisms are also capable of activating the chemical defenses.
Project description:High salinity is one of the most serious threats to crop production. To 1 better understand the molecular basis of plant responses to salt stress, we combined suppression subtractive hybridization (SSH) and microarray approaches to identify the potential important or novel genes involved in salt tolerance. First, SSH libraries were constructed for two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt tolerant cultivar, and ZS-5, a salt sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon salt treatment at various time points compared to the corresponding non-treatment controls. A totalof 201 non-redundant genes differentially expressed upon 30 min of salt stress treatment either in LA2711 or ZS-5 were identified from microarray analysis, most of which were not previously associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants. Keywords: gene expression, genotype, microarray, salt stress, SSH, tomato Overall design: tomato seedling from two different genotypes, LA2711, a salt tolerant cultivar and ZS-5, a salt sensitive cultivar were treated with salt stress for 30 min, 2 hr and 6 hr. Total RNA extracted from root tissue of each treated plant was hybridized to the array together with RNA from control plant. Each hybridization was performed in duplicate by dye swap.
Project description:Beneficial fungi in the genus Trichoderma are among the most widespread biocontrol agents of plant pathogens. Their role in triggering plant defenses against pathogens has been intensely investigated, while, in contrast, very limited information is available on induced barriers active against insects. The growing experimental evidence on this latter topic looks promising, and paves the way toward the development of Trichoderma strains and/or consortia active against multiple targets. However, the predictability and reproducibility of the effects that these beneficial fungi is still somewhat limited by the lack of an in-depth understanding of the molecular mechanisms underlying the specificity of their interaction with different crop varieties, and on how the environmental factors modulate this interaction. To fill this research gap, here we studied the transcriptome changes in tomato plants (cultivar "Dwarf San Marzano") induced by Trichoderma harzianum (strain T22) colonization and subsequent infestation by the aphid Macrosiphum euphorbiae. A wide transcriptome reprogramming, related to metabolic processes, regulation of gene expression and defense responses, was induced both by separate experimental treatments, which showed a synergistic interaction when concurrently applied. The most evident expression changes of defense genes were associated with the multitrophic interaction Trichoderma-tomato-aphid. Early and late genes involved in direct defense against insects were induced (i.e., peroxidase, GST, kinases and polyphenol oxidase, miraculin, chitinase), along with indirect defense genes, such as sesquiterpene synthase and geranylgeranyl phosphate synthase. Targeted and untargeted semi-polar metabolome analysis revealed a wide metabolome alteration showing an increased accumulation of isoprenoids in Trichoderma treated plants. The wide array of transcriptomic and metabolomics changes nicely fit with the higher mortality of aphids when feeding on Trichoderma treated plants, herein reported, and with the previously observed attractiveness of these latter toward the aphid parasitoid Aphidius ervi. Moreover, Trichoderma treated plants showed the over-expression of transcripts coding for several families of defense-related transcription factors (bZIP, MYB, NAC, AP2-ERF, WRKY), suggesting that the fungus contributes to the priming of plant responses against pest insects. Collectively, our data indicate that Trichoderma treatment of tomato plants induces transcriptomic and metabolomic changes, which underpin both direct and indirect defense responses.
Project description:Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato <i>eIF4E</i> genes can confer resistance to viruses, particularly members of the <i>Potyvirus</i> genus. Here, we artificially mutated the <i>eIF4E1</i> gene on chromosome 3 of a commercial cultivar of tomato (<i>Solanum lycopersicum</i> L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus <i>Potyvirus</i>) yielded a significant reduction in susceptibility to the N strain (PVY<sup>N</sup>), but not to the ordinary strain (PVY<sup>O</sup>), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus <i>Cucumovirus</i>). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of <i>eIF4E</i> expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited <i>eIF4E</i> alleles confer an alternative way to manage CMV in tomato fields.
Project description:To unravel the molecular mechanisms of drought responses in tomato, gene expression profiles of two drought-tolerant lines identified from a population of Solanum pennellii introgression lines, and the recurrent parent S. lycopersicum cv. M82, a drought-sensitive cultivar, were investigated under drought stress using tomato microarrays. Around 400 genes identified were responsive to drought stress only in the drought-tolerant lines. These changes in genes expression are most likely caused by the two inserted chromosome segments of S. pennellii, which possibly contain drought-tolerance quantitative trait loci (QTLs). Among these genes are a number of transcription factors and signalling proteins which could be global regulators involved in the tomato responses to drought stress. Genes involved in organism growth and development processes were also specifically regulated by drought stress, including those controlling cell wall structure, wax biosynthesis, and plant height. Moreover, key enzymes in the pathways of gluconeogenesis (fructose-bisphosphate aldolase), purine and pyrimidine nucleotide biosynthesis (adenylate kinase), tryptophan degradation (aldehyde oxidase), starch degradation (beta-amylase), methionine biosynthesis (cystathionine beta-lyase), and the removal of superoxide radicals (catalase) were also specifically affected by drought stress. These results indicated that tomato plants could adapt to water-deficit conditions through decreasing energy dissipation, increasing ATP energy provision, and reducing oxidative damage. The drought-responsive genes identified in this study could provide further information for understanding the mechanisms of drought tolerance in tomato.
Project description:Grafting of elite cultivars onto tolerant rootstocks is an advanced strategy to increase tomato tolerance to sub-optimal temperature. However, a detailed understanding of adaptive mechanisms to sub-optimal temperature in rootstocks and scions of grafting combinations on a physiological and molecular level is lacking. Here, the commercial cultivar Kommeet was grafted either onto 'Moneymaker' (sensitive) or onto the line accession LA 1777 of Solanum habrochaites (tolerant). Grafted plants were grown in NFT-system at either optimal (25°C) or sub-optimal (15°C) temperatures in the root environment with optimal air temperature (25°C) for 22 days. Grafting onto the differently tolerant rootstocks caused differences in shoot fresh and dry weight, total leaf area and dry matter content of roots, in stomatal conductance and intercellular CO2 and guaiacol peroxidase activity but not in net photosynthesis, sugar, starch and amino acid content, lipid peroxidation and antioxidant enzyme activity. In leaves, comparative transcriptome analysis identified 361 differentially expressed genes (DEG) responding to sub-optimal root temperature when 'Kommeet' was grafted onto the sensitive but no when grafted onto the tolerant rootstock. 1509 and 2036 DEG responding to sub-optimal temperature were identified in LA 1777 and 'Moneymaker' rootstocks, respectively. In tolerant rootstocks down-regulated genes were enriched in main stress-responsive functional categories and up-regulated genes in cellulose synthesis suggesting that cellulose synthesis may be one of the main adaptation mechanisms to long-term sub-optimal temperature. Down-regulated genes of the sensitive rootstock showed a similar response, but functional categories of up-regulated genes pointed to induced stress responses. Rootstocks of the sensitive cultivar Moneymaker showed in addition an enrichment of up-regulated genes in the functional categories fatty acid desaturation, phenylpropanoids, biotic stress, cytochrome P450 and protein degradation, indicating that the sensitive cultivar showed more transcriptional adaptation to low temperature than the tolerant cultivar that did not show these changes. Mainly defense-related genes were highly differentially expressed between the tolerant and sensitive rootstock genotypes under sub-optimal temperature in the root environment. These results provide new insights into the molecular mechanisms of long-term sub-optimal temperature tolerance of tomato.