Cu-Electrocatalytic Diazidation of Alkenes at ppm Catalyst Loading.

The 1,2-diamine motif is prevalent in natural products, small-molecule pharmaceuticals, and catalysts for asymmetric synthesis. Transition metal catalyzed alkene diazidation has evolved to be an attractive strategy to access vicinal primary diamines but remains challenging, especially for practical applications, due to the restriction to a certain type of olefins, the frequent use of chemical oxidants, and the requirement for high loadings of metal catalysts (1 mol % or above). Herein we report a scalable Cu-electrocatalytic alkene diazidation reaction with 0.02 mol % (200 ppm) of copper(II) acetylacetonate as the precatalyst without exogenous ligands. In addition to its use of low catalyst loading, the electrocatalytic method is scalable, compatible with a broad range of functional groups, and applicable to the diazidation of α,ß-unsaturated carbonyl compounds and mono-, di-, tri-, and tetrasubstituted unactivated alkenes.

CRISPR-Cas9-mediated mutagenesis of the SlSRM1-like gene leads to abnormal leaf development in tomatoes.

BACKGROUND: Leaves, which are the most important organs of plants, can not only fix carbon sources through photosynthesis, but also absorb nutrients through transpiration. Leaf development directly determines the growth, flowering and fruiting of plants. There are many factors that affect leaf development, such as the growth environment, gene expression, and hormone synthesis. In this study, tomatoes were used to study the role of the transcription factor Solanum lycopersicum salt-related MYB1-like (SlSRM1-like) in the development of tomato leaves. RESULTS: Loss-of-function of the SlSRM1-like gene mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) resulted in abnormal tomato leaf morphology, including thinner leaves, wrinkled edges, raised veins, disordered edge veins, and left and right asymmetry. An analysis of the transcription levels of genes related to leaf development revealed that the expression of these genes was significantly altered in the SlSRM1-like mutants (SlSRM1-like-Ms). Moreover, the SlSRM1-like gene was expressed at higher transcription levels in young tissues than in old tissues, and its expression was also induced in response to auxin. In addition, the transcription levels of genes related to the auxin pathway, which regulates tomato growth and development, were severely affected in the SlSRM1-like-Ms. Therefore, it is hypothesized that the SlSRM1-like gene functions in the regulation of tomato leaf development through the auxin-related pathway. CONCLUSIONS: In this study, we successfully knocked out the SlSRM1-like gene in the tomato variety Ailsa Craig using CRISPR technology and found that knockout of the SlSRM1-like gene resulted in abnormal development of tomato leaves. Further research indicated that SlSRM1-like regulated tomato leaf development through auxin-related pathways. The results provide an important reference for the functional study of other SRM1-like genes in plants and provide new insights into the regulation of leaf development in tomato and other plants.

Genome-wide identification of Tomato Golden 2-Like transcription factors and abiotic stress related members screening.

BACKGROUND: Golden 2-Like (G2-like) transcription factors play an important role in plant development. However, the roles of these G2-like regulatory genes in response to abiotic stresses in tomato are not well understood. RESULTS: In this study, we identified 66 putative G2-like genes in tomato (Solanum lycopersicum) and classified them into 5 groups (I to V) according to gene structure, motif composition and phylogenetic analysis. The G2-like genes were unevenly distributed across all 12 chromosomes. There were nine pairs of duplicated gene segments and four tandem duplicated SlGlk genes. Analysis of the cis-regulatory elements (CREs) showed that the promoter regions of SlGlks contain many kinds of stress- and hormone-related CREs. Based on RNA-seq, SlGlks were expressed in response to three abiotic stresses. Thirty-six differentially expressed SlGlks were identified; these genes have multiple functions according to Gene Ontology (GO) analysis and are enriched mainly in the zeatin biosynthesis pathway. Further studies exhibited that silencing SlGlk16 in tomato would reduce drought stress tolerance by earlier wilted, lower superoxide dismutase (SOD), peroxidase (POD) activities, less Pro contents and more MDA contents. CONCLUSIONS: Overall, the results of this study provide comprehensive information on G2-like transcription factors and G2-like genes that may be expressed in response to abiotic stresses.

The Sm gene conferring resistance to gray leaf spot disease encodes an NBS-LRR (nucleotide-binding site-leucine-rich repeat) plant resistance protein in tomato.

KEY MESSAGES: Gray leaf spot (GLS) resistance in tomato is controlled by one major dominant locus, Sm. Sm was fine mapped, and the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene Solyc11g020100 was identified as a candidate gene for Sm. Further functional analysis indicated that this gene confers high resistance to Stemphylium lycopersici in tomato. Tomato (Solanum Lycopersicum) is widely consumed and cultivated in the world. Gray leaf spot (GLS), caused by Stemphylium lycopersici (S. lycopersici), is one of the most devastating diseases in tomato production. To date, only one resistance gene, Sm, which confers high resistance against GLS disease, has been identified in the wild tomato species Solanum pimpinellifolium. This resistance locus (comprising the Sm gene) has been transferred into the cultivated variety 'Motelle'. Although several studies have reported the mapping of the Sm gene, it has not been cloned, limiting the utilization in tomato breeding. Here, we cloned Sm using a map-based cloning strategy. The Sm gene was mapped in a region of 160 kb at chromosome 11 between two markers, namely, M390 and M410, by using an F2 population from a cross between the resistant cultivar 'Motelle' (Mt) and susceptible line 'Moneymaker' (Mm). Three clustered NBS-LRR (nucleotide-binding site-leucine-rich repeat) resistance genes, namely, Solyc11g020080 (R1), Solyc11g020090 (R2), and Solyc11g020100 (R3) were identified in this interval. Nonsynonymous SNPs were identified in only the open reading frame (ORF) of R3, suggesting it as a strong candidate for the Sm gene. Furthermore, gene silencing of R3 abolished the high resistance to S. lycopersici in Motelle, demonstrating that this gene confers high resistance to S. lycopersici. The cloning of Sm may speed up its utilization for breeding resistant tomato varieties and represents an important step forward in our understanding of the mechanism underlying the resistance to GLS.

Transcriptome Analysis Reveals the Response Mechanism of Frl-Mediated Resistance to Fusarium oxysporum f. sp. radicis-lycopersici (FORL) Infection in Tomato.

Tomato Fusarium crown and root rot (FCRR) is an extremely destructive soil-borne disease. To date, studies have shown that only plants with tomato mosaic virus (TMV) resistance exhibit similar resistance to tomato Fusarium oxysporum f. sp. radicis-lycopersici (FORL) and have identified a single relevant gene, Frl, in Peruvian tomato. Due to the relative lack of research on FCRR disease-resistance genes in China and elsewhere, transcriptome data for FORL-resistant (cv. '19912') and FORL-susceptible (cv. 'Moneymaker') tomato cultivars were analysed for the first time in this study. The number of differentially expressed genes (DEGs) was higher in Moneymaker than in 19912, and 189 DEGs in the 'plant-pathogen interaction' pathway were subjected to GO and KEGG enrichment analyses. MAPK and WRKY genes were enriched in major metabolic pathways related to plant disease resistance; thus, we focused on these two gene families. In the early stage of tomato infection, the content of JA and SA increased, but the change in JA was more obvious. Fourteen genes were selected for confirmation of their differential expression levels by qRT-PCR. This study provides a series of novel disease resistance resources for tomato breeding and genetic resources for screening and cloning FORL resistance genes.

Transcriptome Analysis to Explore the Cause of the Formation of Different Inflorescences in Tomato.

The number of inflorescence branches is an important agronomic character of tomato. The meristem differentiation and development pattern of tomato inflorescence is complex and its regulation mechanism is very different from those of other model plants. Therefore, in order to explore the cause of tomato inflorescence branching, transcriptome analysis was conducted on two kinds of tomato inflorescences (single racemes and compound inflorescences). According to the transcriptome data analysis, there were many DEGs of tomato inflorescences at early, middle, and late stages. Then, GO and KEGG enrichments of DEGs were performed. DEGs are mainly enriched in metabolic pathways, biohormone signaling, and cell cycle pathways. According to previous studies, DEGs were mainly enriched in metabolic pathways, and FALSIFLORA (FA) and ANANTHA (AN) genes were the most notable of 41 DEGs related to inflorescence branching. This study not only provides a theoretical basis for understanding inflorescence branching, but also provides a new idea for the follow-up study of inflorescence.

Transcriptome Analysis of the Cf-13-Mediated Hypersensitive Response of Tomato to Cladosporium fulvum Infection.

Tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum) is one of the most common diseases affecting greenhouse tomato production. Cf proteins can recognize corresponding AVR proteins produced by C. fulvum, and Cf genes are associated with leaf mold resistance. Given that there are many physiological races of C. fulvum and that these races rapidly mutate, resistance to common Cf genes (such as Cf-2, Cf-4, Cf-5, and Cf-9) has decreased. In the field, Ont7813 plants (carrying the Cf-13 gene) show effective resistance to C. fulvum; thus, these plants could be used as new, disease-resistant materials. To explore the mechanism of the Cf-13-mediated resistance response, transcriptome sequencing was performed on three replicates each of Ont7813 (Cf-13) and Moneymaker (MM; carrying the Cf-0 gene) at 0, 9, and 15 days after inoculation (dai) for a total of 18 samples. In total, 943 genes were differentially expressed, specifically in the Ont7813 response process as compared to the Moneymaker response process. Gene ontology (GO) classification of these 943 differentially expressed genes (DEGs) showed that GO terms, including "hydrogen peroxide metabolic process (GO_Process)", "secondary active transmembrane transporter activity (GO_Function)", and "mismatch repair complex (GO_Component)", which were the same as 11 other GO terms, were significantly enriched. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many key regulatory genes of the Cf-13-mediated resistance response processes were involved in the "plant hormone signal transduction" pathway, the "plant-pathogen interaction" pathway, and the "MAPK signaling pathway-plant" pathway. Moreover, during C. fulvum infection, jasmonic acid (JA) and salicylic acid (SA) contents significantly increased in Ont7813 at the early stage. These results lay a vital foundation for further understanding the molecular mechanism of the Cf-13 gene in response to C. fulvum infection.

Transcriptome Analysis and Screening of Genes Associated with Flower Size in Tomato (Solanum lycopersicum).

Flower development is not only an important way for tomato reproduction but also an important guarantee for tomato fruit production. Although more and more attention has been paid to the study of flower development, there are few studies on the molecular mechanism and gene expression level of tomato flower development. In this study, RNA-seq analysis was performed on two stages of tomato flower development using the Illumina sequencing platform. A total of 8536 DEGs were obtained by sequencing, including 3873 upregulated DEGs and 4663 down-regulated DEGs. These differentially expressed genes are related to plant hormone signaling, starch and sucrose metabolism. The pathways such as pentose, glucuronate interconversion, and Phenylpropanoid biosynthesis are closely related and mainly involved in plant cellular and metabolic processes. According to the enrichment analysis results of DEGs, active energy metabolism can be inferred during flower development, indicating that flower development requires a large amount of energy and material supply. In addition, some plant hormones, such as GA, may also have effects on flower development. Combined with previous studies, the expression levels of Solyc02g087860 and three of bZIPs were significantly increased in the full flowering stage compared with the flower bud stage, indicating that these genes may be closely related to flower development. These genes were previously reported in Arabidopsis but not in tomatoes. Our next work will conduct a detailed functional analysis of the identified bZIP family genes to characterize their association with tomato flower size. This study will provide new genetic resources for flower formation and provide a basis for tomato yield breeding.

Genome-wide identification and functional analysis of the ERF2 gene family in response to disease resistance against Stemphylium lycopersici in tomato.

BACKGROUND: APETALA2/ethylene responsive factor (AP2/ERF) transcription factors are a plant-specific family of transcription factors and one of the largest families of transcription factors. Ethylene response factors (ERF) regulate plant growth, development, and responses to biotic and abiotic stress. In a previous study, the ERF2 gene was significantly upregulated in both resistant and susceptible tomato cultivars in response to Stemphylium lycopersici. The main purpose of this study was to systematically analyze the ERF family and to explore the mechanism of ERF2 in tomato plants resisting pathogen infection by the Virus-induced Gene Silencing technique. RESULTS: In this experiment, 134 ERF genes were explored and subjected to bioinformatic analysis and divided into twelve groups. The spatiotemporal expression characteristics of ERF transcription factor gene family in tomato were diverse. Combined with RNA-seq, we found that the expression of 18 ERF transcription factors increased after inoculation with S. lycopersici. In ERF2-silenced plants, the susceptible phenotype was observed after inoculation with S. lycopersici. The hypersensitive response and ROS production were decreased in the ERF2-silenced plants. Physiological analyses showed that the superoxide dismutase, peroxidase and catalase activities were lower in ERF2-silenced plants than in control plants, and the SA and JA contents were lower in ERF2-silenced plants than in control plants after inoculation with S. lycopersici. Furthermore, the results indicated that ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance. CONCLUSIONS: In this study, we identified and analyzed members of the tomato ERF family by bioinformatics methods and classified, described and analyzed these genes. Subsequently, we used VIGS technology to significantly reduce the expression of ERF2 in tomatoes. The results showed that ERF2 had a positive effect on tomato resistance to S. lycopersici. Interestingly, ERF2 played a key role in multiple SA, JA and ROS signaling pathways to confer resistance to invasion by S. lycopersici. In addition, ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance to S. lycopersici. In summary, this study provides gene resources for breeding for disease resistance in tomato.

The α-Subunit of the Chloroplast ATP Synthase of Tomato Reinforces Resistance to Gray Mold and Broad-Spectrum Resistance in Transgenic Tobacco.

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed that the cpATPase CF1 α subunit (AtpA) is a key protein involved in Clonostachys rosea-induced resistance to the fungus Botrytis cinerea in tomato. Here, we show that expression of the tomato atpA gene was upregulated by B. cinerea and Clonostachys rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, atpA-overexpressing tobacco showed increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response-like reactions, balanced reactive oxygen species, alleviated damage to the chloroplast ultrastructure of leaf cells, elevated levels of ATP content and cpATPase activity, and enhanced expression of genes related to carbon metabolism, photosynthesis, and defense. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after inoculation with B. cinerea. In addition, overexpression of atpA conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, AtpA is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection and provides broad-spectrum resistance in plants in the process.

CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans.

Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.

Comparative Genome Analysis of Genes Regulating Compound Inflorescences in Tomato.

Inflorescences are the main factor affecting fruit yield. The quantity and quality of inflorescences are closely related to fruit quality and yield. The presence of compound inflorescences in cherry tomatoes is well established, and it has been discovered by chance that compound racemes also exist in tomatoes. To explore the formation of compound inflorescences in tomato, transcriptome sequencing was performed on Moneymaker (MM) and Compound Inflorescence (CI) plants. In-florescences were collected in three periods (early, middle and late) in three replicates, for a total of 18 samples. Data analysis showed that the DEGs were most enriched in metabolic pathways and plant hormone signal transduction pathways. The DEGs were also enriched in the cell cycle pathway, photosynthesis pathway, carbon metabolism pathway and circadian rhythm pathway. We found that the FALSIFLORA (FA), COMPOUND INFLORESCENCE (S) and ANANTHA (AN) genes were involved in compound inflorescence development, not only revealing novel genes but also providing a rich theoretical basis for compound inflorescence development.

Genome-Wide Identification, Characterization and Expression Analysis of the JAZ Gene Family in Resistance to Gray Leaf Spots in Tomato.

The plant disease resistance system involves a very complex regulatory network in which jasmonates play a key role in response to external biotic or abiotic stresses. As inhibitors of the jasmonic acid (JA) signaling pathway, JASMONATE ZIM domain (JAZ) proteins have been identified in many plant species, and their functions are gradually being clarified. In this study, 26 JAZ genes were identified in tomato. The physical and chemical properties, predicted subcellular localization, gene structure, cis-acting elements, and interspecies collinearity of 26 SlJAZ genes were subsequently analyzed. RNA-seq data combined with qRT-PCR analysis data showed that the expression of most SlJAZ genes were induced in response to Stemphylium lycopersici, methyl jasmonate (MeJA) and salicylic acid (SA). Tobacco rattle virus RNA2-based VIGS vector (TRV2)-SlJAZ25 plants were more resistant to tomato gray leaf spots than TRV2-00 plants. Therefore, we speculated that SlJAZ25 played a negative regulatory role in tomato resistance to gray leaf spots. Based on combining the results of previous studies and those of our experiments, we speculated that SlJAZ25 might be closely related to JA and SA hormone regulation. SlJAZ25 interacted with SlJAR1, SlCOI1, SlMYC2, and other resistance-related genes to form a regulatory network, and these genes played an important role in the regulation of tomato gray leaf spots. The subcellular localization results showed that the SlJAZ25 gene was located in the nucleus. Overall, this study is the first to identify and analyze JAZ family genes in tomato via bioinformatics approaches, clarifying the regulatory role of SlJAZ25 genes in tomato resistance to gray leaf spots and providing new ideas for improving plant disease resistance.

Transcriptome Analysis of Flower Development and Mining of Genes Related to Flowering Time in Tomato (Solanum lycopersicum).

Flowering is a morphogenetic process in which angiosperms shift from vegetative growth to reproductive growth. Flowering time has a strong influence on fruit growth, which is closely related to productivity. Therefore, research on crop flowering time is particularly important. To better understand the flowering period of the tomato, we performed transcriptome sequencing of early flower buds and flowers during the extension period in the later-flowering "Moneymaker" material and the earlier-flowering "20965" homozygous inbred line, and we analyzed the obtained data. At least 43.92 million clean reads were obtained from 12 datasets, and the similarity with the tomato internal reference genome was 92.86-94.57%. Based on gene expression and background annotations, 49 candidate genes related to flowering time and flower development were initially screened, among which the greatest number belong to the photoperiod pathway. According to the expression pattern of candidate genes, the cause of early flowering of "20965" is predicted. The modes of action of the differentially expressed genes were classified, and the results show that they are closely related to hormone regulation and participated in a variety of life activities in crops. The candidate genes we screened and the analysis of their expression patterns provide a basis for future functional verification, helping to explore the molecular mechanism of tomato flowering time more comprehensively.

Transcriptomic profiling of Solanum peruvianum LA3858 revealed a Mi-3-mediated hypersensitive response to Meloidogyne incognita.

BACKGROUND: The Mi-1 gene was the first identified and cloned gene that provides resistance to root-knot nematodes (RKNs) in cultivated tomato. However, owing to its temperature sensitivity, this gene does not meet the need for breeding disease-resistant plants that grow under high temperature. In this study, Mi-3 was isolated from the wild species PI 126443 (LA3858) and was shown to display heat-stable resistance to RKNs. However, the mechanism that regulates this resistance remains unknown. RESULTS: In this study, 4760, 1024 and 137 differentially expressed genes (DEGs) were enriched on the basis of pairwise comparisons (34 °C vs. 25 °C) at 0 (before inoculation), 3 and 6 days post-inoculation (dpi), respectively. A total of 7035 DEGs were identified from line LA3858 in the respective groups under the different soil temperature treatments. At 3 dpi, most DEGs were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to plant biotic responses, such as "plant-pathogen interaction" and "plant hormone signal transduction". Significantly enriched DEGs were found to encode key proteins such as R proteins and heat-shock proteins (HSPs). Moreover, other DEGs were found to participate in Ca2+ signal transduction; the production of ROS; DEGs encoding transcription factors (TFs) from the bHLH, TGA, ERF, heat-shock transcription factor (HSF) and WRKY families were highly expressed, which contribute to be involved into the formation of phytohormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), the expression of most was upregulated at 3 dpi at the 25 °C soil temperature compared with the 34 °C soil temperature. CONCLUSION: Taken together, the results of our study revealed reliable candidate genes from wild materials LA3858, that are related to Mi-3-mediate resistance to Meloidogyne incognita. A large number of vital pathways and DEGs were expressed specifically in accession LA3858 grown at 34 °C and 25 °C soil temperatures at 3 dpi. Upon infection by RKNs, pattern-recognition receptors (PRRs) specifically recognized conserved pathogen-associated molecular patterns (PAMPs) as a result of pathogen-triggered immunity (PTI), and the downstream defensive signal transduction pathway was likely activated through Ca2+ signal channels. The expression of various TFs was induced to synthesize phytohormones and activate R proteins related to resistance, resulting in the development of effector-triggered immunity (ETI). Last, a hypersensitive response in the roots occurred, which was probably induced by the accumulation of ROS.

Functional analysis of the SlERF01 gene in disease resistance to S. lycopersici.

BACKGROUND: Tomato gray leaf spot caused by Stemphylium lycopersici (S. lycopersici) is a serious disease that can severely hinder tomato production. To date, only Sm has been reported to provide resistance against this disease, and the molecular mechanism underlying resistance to this disease in tomato remains unclear. To better understand the mechanism of tomato resistance to S. lycopersici, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based analysis, physiological indexes, microscopy observations and transgenic technology were used in this study. RESULTS: Our results showed that the expression of SlERF01 was strongly induced by S. lycopersici and by exogenous applications of the hormones salicylic acid (SA) and jasmonic acid (JA). Furthermore, overexpression of SlERF01 enhanced the hypersensitive response (HR) to S. lycopersici and elevated the expression of defense genes in tomato. Furthermore, the accumulation of lignin, callose and hydrogen peroxide (H2O2) increased in the transgenic lines after inoculation with S. lycopersici. Taken together, our results showed that SlERF01 played an indispensable role in multiple SA, JA and reactive oxygen species (ROS) signaling pathways to provide resistance to S. lycopersici invasion. Our findings also indicated that SlERF01 could activate the expression of the PR1 gene and enhance resistance to S. lycopersici. CONCLUSIONS: We identified the SlERF01 gene, which encodes a novel tomato AP2/ERF transcription factor (TF). Functional analysis revealed that SlERF01 positively regulates tomato resistance to S. lycopersici. Our findings indicate that SlERF01 plays a key role in multiple SA, JA and ROS signaling pathways to provide resistance to invasion by S. lycopersici. The findings of this study not only help to better understand the mechanisms of response to pathogens but also enable targeted breeding strategies for tomato resistance to S. lycopersici.

Comparative transcriptome analysis reveals the response mechanism of Cf-16-mediated resistance to Cladosporium fulvum infection in tomato.

BACKGROUND: Leaf mold disease caused by Cladosporium fulvum is a serious threat affecting the global production of tomato. Cf genes are associated with leaf mold resistance, including Cf-16, which confers effective resistance to leaf mold in tomato. However, the molecular mechanism of the Cf-16-mediated resistance response is largely unknown. RESULTS: We performed a comparative transcriptome analysis of C. fulvum-resistant (cv. Ontario7816) and C. fulvum-susceptible (cv. Moneymaker) tomato cultivars to identify differentially expressed genes (DEGs) at 4 and 8 days post inoculation (dpi) with C. fulvum. In total, 1588 and 939 more DEGs were found in Cf-16 tomato than in Moneymaker at 4 and 8 dpi, respectively. Additionally, 1350 DEGs were shared between the 4- and 8-dpi Cf-16 groups, suggesting the existence of common core DEGs in response to C. fulvum infection. The up-regulated DEGs in Cf-16 tomato were primarily associated with defense processes and phytohormone signaling, including salicylic acid (SA) and jasmonic acid (JA). Moreover, SA and JA levels were significantly increased in Cf-16 tomato at the early stages of C. fulvum infection. Contrary to the previous study, the number of up-regulated genes in Cf-16 compared to Cf-10 and Cf-12 tomatoes was significantly higher at the early stages of C. fulvum infection. CONCLUSION: Our results provide new insight into the Cf-mediated mechanism of resistance to C. fulvum, especially the unique characteristics of Cf-16 tomato in response to this fungus.

Molecular mapping of the Cf-10 gene by combining SNP/InDel-index and linkage analysis in tomato (Solanum lycopersicum).

BACKGROUND: Leaf mold, one of the major diseases of tomato caused by Cladosporium fulvum (C. fulvum), can dramatically reduce the yield and cause multimillion dollar losses annually worldwide. Mapping the resistance genes (R genes) of C. fulvum and devising MAS based strategies for breeding new cultivars is an effective approach to improve the resistance in tomato. Up to now, many C. fulvum genes or QTLs have been mapped using different genetic materials, but few studies focused on Cf-10 gene positioning. RESULTS: In this study, we investigated the genetic rules for Cf-10 and used a novel combinatorial strategy to rapidly map the Cf-10 gene. Initially, the performance of F1, F2 and BC1F1 individuals after infection, demonstrated that the resistance against C. fulvum was controlled by a single dominant gene. Two pools of resistant and susceptible individuals from F2 population were investigated, using mapping by sequencing approach and Cf-10 was found to be localized to 3.35 Mb and 3.74 Mb on chromosome 1, employing SNP/InDel index methods, respectively. After accounting for overlapping regions, these two algorithms yielded a total length of 3.29 Mb, narrowing down the target region. We further developed five serviceable KASP markers for this region based on sequencing data and conducted local QTL mapping using individuals from the F2 population, except for mapping by sequencing as mentioned above. Finally Cf-10 gene was mapped spanning a region of 790 kb, where only one gene (Solyc01g007130.3) was annotated as probable receptor protein kinase TMK1 with a LRR motif, a common R gene characteristic. The RT-qPCR analysis further confirmed the localization and the relative expression of Solyc01g007130.3 in Ontario 792 and was found to be significantly higher than that in Moneymaker at 9 dpi and 12 dpi, respectively. CONCLUSION: This study proposed a novel combinatorial strategy by combining SNP-index, InDel-index analyses and local QTL mapping using KASP genotyping approach to rapidly map genes responsible for specific traits and provided a robust base for cloning the Cf-10 gene. Furthermore, these analyses suggest that Solyc01g007130.3 is a potential candidate to be regarded as Cf-10 gene.

Transcriptome profiling reveals the response process of tomato carrying Cf-19 and Cladosporium fulvum interaction.

BACKGROUND: During tomato cultivation, tomato leaf mould is a common disease caused by Cladosporium fulvum (C. fulvum). By encoding Cf proteins, which can recognize corresponding AVR proteins produced by C. fulvum, Cf genes provide resistance to C. fulvum, and the resistance response patterns mediated by different Cf genes are not identical. Plants carrying the Cf-19 gene show effective resistance to C. fulvum in the field and can be used as new resistant materials in breeding. In this study, to identify key regulatory genes related to resistance and to understand the resistance response process in tomato plants carrying Cf-19, RNA sequencing (RNA-seq) was used to analyse the differences between the response of resistant plants (CGN18423, carrying the Cf-19 gene) and susceptible plants (Moneymaker (MM), carrying the Cf-0 gene) at 0, 7 and 20 days after inoculation (dai). RESULTS: A total of 418 differentially expressed genes (DEGs) were identified specifically in the CGN18423 response process. Gene Ontology (GO) analysis revealed that GO terms including "plasma membrane (GO_Component)", "histidine decarboxylase activity (GO_Function)", and "carboxylic acid metabolic process (GO_Process)", as well as other 10 GO terms, were significantly enriched. The "plant hormone signal transduction" pathway, which was unique to CGN18423 in the 0-7 dai comparison, was identified. Moreover, ten key regulatory points were screened from the "plant hormone signal transduction" pathway and the "plant pathogen interaction" pathway. Hormone content measurements revealed that the salicylic acid (SA) contents increased and peaked at 7 dai, after which the contents deceased and reached minimum values in both CGN18423 and MM plants at 20 dai. The jasmonic acid (JA) content increased to a very high level at 7 dai but then decreased to nearly the initial level at 20 dai in CGN18423, while it continued to increase slightly during the whole process from 0 to 20 dai in MM. CONCLUSIONS: The initial responses are very different between the resistant and susceptible plants. The "plant hormone signal transduction" pathway is important for the formation of Cf-19-mediated immunity. In addition, both JA and SA play roles in regulating the Cf-19-dependent resistance response.

Physiological and RNA-seq analyses provide insights into the response mechanism of the Cf-10-mediated resistance to Cladosporium fulvum infection in tomato.

KEY MESSAGE: Based on the physiological and RNA-seq analysis, some progress has been made in elucidating the Cf-10-mediated resistance responses to C. fulvum infection in tomato. GO and KEGG enrichment analysis revealed that the DEGs were significantly associated with defense-signaling pathways like oxidation-reduction processes, oxidoreductase activity and plant hormone signal transduction. Leaf mold, caused by the fungus Cladosporium fulvum, is one of the most common diseases affecting tomatoes worldwide. Cf series genes including Cf-2, Cf-4, Cf-5, Cf-9 and Cf-10 play very important roles in resisting tomato leaf mold. Understanding the molecular mechanism of Cf gene-mediated resistance is thus the key to facilitating genetic engineering of resistance to C. fulvum infection. Progress has been made in elucidating two Cf genes, Cf -19 and Cf -12, and how they mediate resistance responses to C. fulvum infection in tomato. However, the mechanism of the Cf-10- mediated resistance response is still unclear. In the present study, RNA-seq was used to analyze changes in the transcriptome at different stages of C. fulvum infection. A total of 2,242 differentially expressed genes (DEGs) responsive to C. fulvum between 0 and 16 days post infection (dpi) were identified, including 1,501 upregulated and 741 downregulated genes. The majority of DEGs were associated with defense-signaling pathways including oxidation-reduction processes, oxidoreductase activity and plant hormone signal transduction. Four DEGs associated with plant-pathogen interaction were uniquely activated in Cf-10 tomato and validated by qRT-PCR. In addition, physiological indicators including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were measured at 0-21 dpi, and hormone expression [Jasmonic acid (JA) and salicylic acid (SA)] was estimated at 0 and 16 dpi to elucidate the mechanism of the Cf-10-mediated resistance response. C. fulvum infection induced the activities of POD, CAT and SOD, and decreased ROS levels. JA was determined to participate in the resistance response to C. fulvum during the initial infection period. The results of this study provide accountable evidence for the physiological and transcriptional regulation of the Cf-10-mediated resistance response to C. fulvum infection, facilitating further understanding of the molecular mechanism of Cf-10-mediated resistance to C. fulvum infection.