Tomato fruit susceptibility to fungal disease is not an inevitable outcome of ripening and can be uncoupled by targeting susceptibility factors
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ABSTRACT: The increased susceptibility of ripe fruit to fungal pathogens poses a substantial threat to crop production and marketability. Here, we coupled transcriptomic analyses with mutant studies to uncover critical genes and processes governing ripening-associated susceptibility in tomato (Solanum lycopersicum) fruit. Using wild-type unripe and ripe fruit inoculated with three fungal pathogens—Botrytis cinerea, Fusarium acuminatum, and Rhizopus stolonifer—we identified common pathogen response genes reliant on chitinases, WRKY transcription factors, and reactive oxygen species detoxification. Interestingly, susceptible ripe fruit demonstrated a more extensive defense response than resistant unripe fruit, indicating that the magnitude and diversity of defense response does not significantly impact the interaction. To tease apart individual features of ripening that may be responsible for susceptibility, we utilized three tomato non-ripening mutants: Cnr, rin and nor. Fruit from these mutants displayed different patterns of susceptibility to fungal infection. Functional analysis of the genes altered during ripening in the susceptible genotypes revealed losses in the maintenance of cellular redox homeostasis. Moreover, jasmonic acid accumulation and signaling coincided with the activation of defenses in resistant fruit. Lastly, based on high gene expression in susceptible fruit, we identified and tested two candidate susceptibility factors, pectate lyase (PL) and polygalacturonase (PG2a). CRISPR-based knockouts of PL, but not PG2a, resulted in more than 50% decrease in the susceptibility of ripe fruit, demonstrating that PL is a major susceptibility factor. Ultimately, this study demonstrates that targeting specific genes that drive susceptibility is a viable strategy to improve resistance of tomato fruit against fungal pathogens.
Project description:Tomato fruit ripening is associated with a dramatic increase in susceptibility to the fungal pathogen Botrytis cinerea, the causal agent of gray mold. Mature green fruit, prior to ripening, are largely resistant to B. cinerea, whereas red fruit, at the end of ripening, are susceptible to B. cinerea infection. We used microarrays to detail the gene expression changes that are induced by B. cinerea when tomato fruit at unripe and ripe stages are infected. Keywords: plant responses to pathogens
Project description:Tomato fruit ripening is associated with a dramatic increase in susceptibility to the fungal pathogen Botrytis cinerea, the causal agent of gray mold. Mature green fruit, prior to ripening, are largely resistant to B. cinerea, whereas red fruit, at the end of ripening, are susceptible to B. cinerea infection. We used microarrays to detail the gene expression changes that are induced by B. cinerea when tomato fruit at unripe and ripe stages are infected. Experiment Overall Design: Tomato fruit at mature green and red ripe stages were wound inoculated with a water suspension of B. cinerea conidia. Twenty four hours post inoculation fruit pericarp and epicarp tissue around and including the inoculation sites was collected and the total RNA extracted. Total RNA was also collected from healthy and mock inoculated fruit.
Project description:Worldwide, 20-25% of all harvested fruit and vegetables are lost annually in the field and throughout the postharvest supply handling chain due to spoilage by fungal pathogens. Most impactful postharvest pathogens exhibit necrotrophic lifestyles, resulting in rotting of the host tissues and complete loss of marketable commodities. Necrotrophic fungi can readily infect ripe fruit leading to the rapid establishment of disease symptoms. However, these pathogens generally fail to infect unripe fruit, or remain quiescent until host and environmental conditions stimulate a successful infection. Current research on necrotrophic infections of fruit was mainly focused on the host by characterizing genetic and physicochemical factors that inhibit or promote the disease. However, the pathogenicity and virulence strategies employed by necrotrophic pathogens in ripe and unripe fruit are mostly understudied. Here, we provide a first comparative transcriptomics study of fungal postharvest pathogens: Botrytis cinerea, Rhizopus stolonifer and Fusarium acuminatum, all of which display necrotrophic behavior when infecting fruit. We de novo assembled and annotated the transcriptomes of R. stolonifer, and F. acuminatum and performed a differential gene expression analysis comparing the three fungal transcriptomes during fruit infection with that of fungal in-vitro growth. Analysis of the differentially expressed genes for enrichment of functional annotations revealed shared strategies of the three fungi during infection of compatible (ripe fruit) and incompatible (unripe fruit) hosts. We furthermore selected candidate genes that are involved in these strategies to characterize their expression during infection of unripe and ripe-like fruit of the non-ripening (nor) tomato mutant, both of which are physiologically and biochemically similar to unripe wildtype fruit. By enabling a better understanding of fungal necrotrophic infection strategies, we move closer to generating accurate models of fruit diseases and development of early detection tools and effective management strategies.
Project description:Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes in order to facilitate infections and promote disease. Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, ELISA-based glycomics profiling of fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectin polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening is dependent on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbcpg1Δbcpg2 lacking two critical pectin degrading enzymes was found to be incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.
Project description:Background: Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during fruit ripening and its control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results: Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes and some of them had their expression validated by qPCR. The transcription profile was then compared with that from ripening tomato and grape. Overall, the transcriptomics analysis revealed many similarities between ripening in papaya and tomato especially with respect to primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicate that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families are involved in the control of papaya ripening and reveal that cell wall-related gene expression in papaya showed similarities to the expression profiles seen in A. thaliana during hypocotyl development. Conclusion: The cross-species array experiment was successful in identifying ripening-related genes in papaya. The data indicated common and diverse elements of transcription control between fruit bearing taxa and has also indicated a possible distinct co-evolutionary mechanism for papaya cell wall disassembling system. The present study represents new topics for future researches that would help complement the structural genomic data provided by the papaya genome, since there is no gene-chip available for this plant organism. Papaya ripe transcriptome was analysed using mRNA extracted from unripe and ripe fruit from 2 replicates. After microarray hybridization in ATH1-121501 chip, data were normalized against data generated by papaya DNA hybridization in another ATH1-121501 chip and analysed using perl algorithms (masks).
Project description:Tomato fruit ripening is under the control of ethylene as well as a group of ethylene-independent transcription factors, including NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN). During ripening, the linear carotene lycopene accumulates at the expense of cyclic carotenoids. Fruit-specific overexpression of LYCOPENE β-CYCLASE (LCYb) under the control of the PHYTOENE DESATURASE (PDS) promoter resulted in increased levels of β-carotene and ABA and in decreased ethylene levels. Genes regulated by ABA, or involved in its synthesis and signaling, were overexpressed, while those associated with ethylene and cell wall remodeling were repressed. In agreement with the transcriptional data, LCYb-overexpressing fruits exhibited increased density of cell wall material containing linear, under-methylated pectins and displayed an array of additional ripening phenotypes, including delayed softening, increased turgor, enhanced shelf life and a thicker cuticle with a higher content of cutin monomers and triterpenoids. The levels of several primary metabolites and phenylpropanoids also changed in the transgenics, which could be attributed to delayed fruit ripening and to ABA respectively. Network correlation analysis suggests that ABA, acting through NOR and RIN, is responsible for many of the above phenotypes. These data reinforce suggestions that ABA plays an important role in tomato fruit ripening and provide clues that fruit b-carotene, acting as a precursor for ABA, actively participates in controlling the ripening process rather than merely being an output thereof. Overexpression of a LCYb gene from Arabidopsis under the control of the ripening-associated PDS promoter leads to ripe tomato fruits accumulating high β-carotene levels. Using several independent transgenic lines, we conducted a system-wide study of the effect of increased β-carotene levels on tomato fruit ripening and shelf life. Our data suggest that β-carotene, acting through ABA, is involved in a regulatory loop within the network controlling tomato fruit ripening.
Project description:Here, we found that the tomato jmjC domain-containing gene SlJMJ6 encodes a histone lysine demethylase that specifically demethylates H3K27 methylation. Overexpression of SlJMJ6 accelerated fruit ripening in tomato, which is associated with the up-regulated expression of a large number of ripening-related genes. Integrated analysis of RNA-seq and ChIP-seq data identified 55 genes that are targeted directly by SlJMJ6 and transcriptionally up-regulated with decreased H3K27m3 in SlJMJ6 overexpressing (SlJMJ6-OE) fruits. A large number of the SlJMJ6-regulated genes are involved in transcription regulation, ethylene biosynthesis, cell wall degradation, pigment biosynthesis, and hormone signaling. Fourteen ripening-related genes including RIN, ACS4, ACO1, PL, TBG4 were confirmed to be directly regulated by SlJMJ6 through removing H3K27me3. Taken together, these results indicated that SlJMJ6 is a ripening prompting H3K27me3 demethylase that activates the expression of the ripening-related genes by modulating H3K27me3, thereby facilitating fruit ripening in tomato. To our knowledge, this is the first report of the involvement of a histone lysine demethylase in the regulation of fruit ripening.
Project description:Here, we found that the tomato jmjC domain-containing gene SlJMJ6 encodes a histone lysine demethylase that specifically demethylates H3K27 methylation. Overexpression of SlJMJ6 accelerated fruit ripening in tomato, which is associated with the up-regulated expression of a large number of ripening-related genes. Integrated analysis of RNA-seq and ChIP-seq data identified 55 genes that are targeted directly by SlJMJ6 and transcriptionally up-regulated with decreased H3K27m3 in SlJMJ6 overexpressing (SlJMJ6-OE) fruits. A large number of the SlJMJ6-regulated genes are involved in transcription regulation, ethylene biosynthesis, cell wall degradation, pigment biosynthesis, and hormone signaling. Fourteen ripening-related genes including RIN, ACS4, ACO1, PL, TBG4 were confirmed to be directly regulated by SlJMJ6 through removing H3K27me3. Taken together, these results indicated that SlJMJ6 is a ripening prompting H3K27me3 demethylase that activates the expression of the ripening-related genes by modulating H3K27me3, thereby facilitating fruit ripening in tomato. To our knowledge, this is the first report of the involvement of a histone lysine demethylase in the regulation of fruit ripening.
Project description:The aim of this study is to generate gene regulatory networks by analyzing Affymetrix GeneChip expression datasets from four different stages of tomato fruit ripening, Breaker (Br), Turning (Tu), Pink (Pk) and Red Ripe (RR) with the LeMoNe algorithm in order to identify co expression modules and their putative regulatory TFs.
Project description:The study investigated protein dynamics throughout fruit developmental and ripening process of blue-colored bilberry. The proteomic approach was applied to study at four different ripening stages, S2-small green fruit, S3- large green fruit, S4- purple ripening fruit, S5- ripe, blue fruit of bilberry. Regulatory network of plant hormones and physiological processes occurring during bilberry fruit ripening was revealed for the first time. The white-colored mutant bilberry, at the ripe stage, was also investigated differences compared to wild, blue-colored berries.