Jun Meng

High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans

AbstractMain conclusionThe characterization and compare expression profiling of the miRNA transcriptome lay a solid foundation for unraveling the complex miRNA-mediated regulatory network in tomato resistance mechanisms against LB.n MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs with 20–24xa0nt. They have been identified in many plants with their diverse regulatory roles in biotic stresses. The knowledge, that miRNAs regulate late blight (LB), caused by Phytophthora infestans, is rather limited. In this study, we used miRNA-Seq to investigate the miRNA expression difference between the tomatoes treated with and without P. infestans. A total of 42,714,516 raw reads were generated from two small RNA libraries by high-throughput sequencing. Finally, 207 known miRNAs and 67 new miRNAs were obtained. The differential expression profile of miRNAs in tomato was further analyzed with twofold change (P value ≤0.01). A total of 70 miRNAs were manifested to change significantly in samples treated with P. infestans, including 50 down-regulated miRNAs and 20 up-regulated miRNAs. Moreover, a total of 73 target genes were acquired for 28 differentially expressed miRNAs by psRNATarget analysis. By enrichment pathway analysis of target genes, plant–pathogen interaction was the most highly relevant pathway which played an important role in disease defense. In addition, 30 miRNAs were selected for qRT-PCR to validate their expression patterns. The expression patterns for targets of miR6027, miR5300, miR476b, miR159a, miR164a and miRn13 were selectively examined, and the results showed that there was a negative correlation on the expression patterns between miRNAs and their targets. The targets have previously been reported to be related with plant immune and involved in plant–pathogen interaction pathway in this study, suggesting these miRNAs might act as regulators in process of tomato resistance against P. infestans. These discoveries will provide us useful information to explain tomato resistance mechanisms against LB.

Comparative transcriptome analysis between resistant and susceptible tomato allows the identification of lncRNA16397 conferring resistance to Phytophthora infestans by co-expressing glutaredoxin.

&NA; The rapid development of omics sequencing technology has facilitated the identification of thousands of long non‐coding (lnc)RNAs in plant species, but the role of lncRNAs in plant–pathogen interactions remains largely unexplored. We used comparative transcriptome analysis of Phytophthora infestans‐resistant and ‐susceptible tomatoes to identify differentially expressed genes (DEGs) and lncRNAs (DELs), and examine lncRNA‐mRNA networks. A total of 1037 DEGs and 688 DELs were identified between P. infestans‐resistant and ‐susceptible tomatoes. The co‐localization networks, including 128 DEGs and 127 DELs, were performed. We found that lncRNA16397 acted as an antisense transcript of SlGRX22 to regulate its expression, and also induced SlGRX21 expression when lncRNA16397 was overexpressed. In addition, disease symptoms and reactive oxygen species (ROS) accumulation in tomatoes overexpressing lncRNA16397 and SpGRX were fewer and lower than those in wild‐type after P. infestans infection. This result suggests that tomato lncRNA16397 induces SlGRX expression to reduce ROS accumulation and alleviate cell membrane injury, resulting in enhanced resistance to P. infestans. Our results provide insight into lncRNAs involved in the response of tomato to P. infestans infection, demonstrate that the lncRNA16397‐GRXs network is an important component of the P. infestans network in tomato, and provide candidates for breeding to enhance biotic stress‐resistance in tomato. Significance Statement There are thousands of lncRNAs in plants, but their role in plant‐pathogen interactions remains largely unexplored. Here, by using comparative transcriptome analysis of Phytophthora infestans‐resistant and susceptible tomato, we show that lncRNA16397 can induce gene expression to reduce ROS accumulation and alleviate cell membrane injury, thus enhancing resistance to P. infestans. We also pinpoint other candidate genes that can be used to breed for enhanced biotic stress‐resistance.

MiR1918 enhances tomato sensitivity to Phytophthora infestans infection

Late blight of tomato is caused by the oomycete pathogen Phytophthora infestans. In our previous work, we identified and characterized a miR1918 in P. infestans (pi-miR1918), and showed that its sequence is similar to the sequence of tomato miR1918 (sly-miR1918). In this study, we used Arabidopsis thaliana pre-miR159a as a backbone to synthesize pi-miR1918 via PCR and mutagenesis. The artificial pi-miR1918 was used to investigate the role of miR1918 in tomato-P. infestans interaction. Trangenic tomato plants that overexpressed the artificial pi-miR1918 displayed more serious disease symptoms than wild-type tomato plants after infection with P. infestans, as shown by increased number of necrotic cells, lesion sizes and number of sporangia per leaf. The target genes of pi-miR1918 and sly-miR1918 were also predicted for tomato and P. infestans, respectively. qPCR analysis of these targets also performed during tomato-P. infestans interaction. The expression of target gene, RING finger were negatively correlated with miR1918 in the all Lines of transgenic tomato plants. In addition, we used the 5′ RACE to determine the cleavage site of miR1918 to RING finger. These results suggested that miR1918 might be involved in the silencing of target genes, thereby enhancing the susceptibility of tomato to P. infestans infection.

Effective enhancement of resistance to Phytophthora infestans by overexpression of miR172a and b in Solanum lycopersicum

AbstractMain conclusionOverexpression of miR172a and b in tomato (Solanum lycopersicum) Zaofen No. 2 increased resistance toPhytophthora infestansinfection by suppressing of an AP2/ERF transcription factor.n The miR172 family has been shown to participate in the growth phase transition, flowering time control, abiotic and biotic stresses by regulating the expression of a small group of AP2/ERF transcription factors. In this study, the precursors of miR172a and b were cloned from tomato, Solanum pimpinellifolium L3708. We used the degradome sequencing to determine the cleavage site of miR172 to a member of the AP2/ERF transcription factor family (Solyc11g072600.1.1). qRT-PCR results showed that the expression of AP2/ERF was negatively correlated with the expression of miR172 in S. pimpinellifolium L3708 infected with Phytophthora infestans. Overexpression of miR172a and b in S. lycopersicum Zaofen No. 2 conferred greater resistance to P. infestans infection, as evidenced by decreased disease index, lesion sizes, and P. infestans abundance. The SOD and POD play important roles in scavenging late massive ROS in plant–pathogen interaction. Malonaldehyde (MDA) is widely recognized as an indicator of lipid peroxidation. Membrane damage in plants can be estimated by measuring leakage of electrolytes, which is evaluated by determining relative electrolyte leakage (REL). Less H2O2 and O2−, higher activities of POD and SOD, less MDA content and REL, and higher chlorophyll content and photosynthetic rate were also shown in transgenic plants after inoculation with P. infestans. Our results constitute the first step towards further investigations into the biological function and molecular mechanism of miR172-mediated silencing of AP2/ERF transcription factors in S. lycopersicum–P. infestans interaction and provide a candidate gene for breeding to enhance biotic stress-resistance in S. lycopersicum.

Transcriptome signatures of tomato leaf induced by Phytophthora infestans and functional identification of transcription factor Sp WRKY3

Key messageSpWRKY3 was identified as a resistance gene to Phytophthora infestans from Solanum pimpinellifolium L3708 and its transgenic tomato showed a significant resistance to P. infestans. This finding reveals the potential application of SpWRKY3 in future molecular breeding.AbstractTranscription factors (TFs) play crucial roles in the plant response to various pathogens. In this present study, we used comparative transcriptome analysis of tomatoes inoculated with and without Phytophthora infestans to identify 1103 differentially expressed genes. Seven enrichment GO terms (level 4) associated with the plant resistance to pathogens were identified. It was found that thirty-five selected TF genes from GO enriched term, sequence-specific DNA binding transcription factor activity (GO: 0003700), were induced by P. infestans. Of these TFs, the accumulation of a homologous gene of WRKY (SpWRKY3) was significantly changed after P. infestans induction, and it was also isolated form P. infestans-resistant tomato, Solanum pimpinellifolium L3708. Overexpression of SpWRKY3 in tomato positively modulated P. infestans defense response as shown by decreased number of necrotic cells, lesion sizes and disease index, while the resistance was impaired after SpWRKY3 silencing. After P. infestans infection, the expression levels of PR genes in transgenic tomato plants overexpressed SpWRKY3 were significantly higher than those in WT, while the number of necrotic cells and the reactive oxygen species (ROS) accumulation were fewer and lower. These results suggest that SpWRKY3 induces PR gene expression and reduces the ROS accumulation to protect against cell membrane injury, leading to enhanced resistance to P. infestans. Our results provide insight into SpWRKY3 as a positive regulator involved in tomato–P. infestans interaction, and its function may enhance tomato resistance to P. infestans.

Characterization and Function of MicroRNA∗s in Plants

MicroRNAs, a group of non-coding RNA molecules, play essential roles in a wide range of cellular processes in different molecules, cells, and organisms. In plants, microRNAs are a class of 20- to 24-nucleotides endogenous small RNAs that repress gene expression. The microRNA guide strand (miRNA) and its complementary strand (miRNA∗) both originate from the miRNA/miRNA∗ duplex. Generally, the guide strands act as post-transcriptional regulators that suppress gene expression by cleaving their target mRNA transcripts, whereas the complementary strands were thought to be degraded as ‘passenger strands.’ However, the complementary strand has been confirmed to possess significant biological functionality in recent reports. In this review, we summarized the binding characteristics of the miRNA∗ strands with ARGONAUTE proteins, their tissue-specific accumulations and their biological functions, illustrating the essential roles of miRNA∗s in biological processes and therefore providing directions for further exploration.

A tomato NBS-LRR gene is positively involved in plant resistance to Phytophthora infestans.

The nucleotide binding sites-leucine-rich repeat (NBS-LRR) genes are key regulatory components of plant to pathogens. Phytophthora infestans-inducible coding sequence encoding an NBS-LRR (SpNBS-LRR) protein in tomato (Solanum pimpinellifolium L3708) was cloned and characterized based on our RNA-Seq data and tomato genome. After sequence analysis, SpNBS-LRR was identified as a hydrophilic protein with no transmembrane topological structure and no signal peptide. SpNBS-LRR had a close genetic relationship to RPS2 of Arabidopsis thaliana by phylogenetic analysis. In addition, SpNBS-LRR gene was mainly expressed in root, with low expression observed in leaf and stem. To further investigate the role of SpNBS-LRR in tomato-P. infestans interaction, SpNBS-LRR was introduced in susceptible tomatoes and three transgenic lines with higher expression level of SpNBS-LRR were selected. These transgenic tomato plants that overexpressed SpNBS-LRR displayed greater resistance than wild-type tomato plants after infection with P. infestans, as shown by decreased disease index, lesion diameters, number of necrotic cells, P. infestans abundance, and higher expression levels of the defense-related genes. This information provides insight into SpNBS-LRR involved in the resistance of tomato to P. infestans infection and candidate for breeding to enhance biotic stress-resistance in tomato.

Tomato MYB49 enhances resistance to Phytophthora infestans and tolerance to water deficit and salt stress

Main conclusionMYB49-overexpressing tomato plants showed significant resistance to Phytophthora infestans and tolerance to drought and salt stresses. This finding reveals the potential application of tomato MYB49 in future molecular breeding.Biotic and abiotic stresses severely reduce the productivity of tomato worldwide. Therefore, it is necessary to find key genes to simultaneously improve plant resistance to pathogens and tolerance to various abiotic stresses. In this study, based on homologous relationships with Arabidopsis R2R3-MYBs (AtMYBs) involved in responses to biotic and abiotic stresses, we identified a total of 24 R2R3-MYB transcription factors in the tomato genome. Among these tomato R2R3-MYBs, MYB49 (Solyc10g008700.1) was clustered into subgroup 11 by phylogenetic analysis, and its expression level was significantly induced after treatment with P. infestans, NaCl and PEG6000. Overexpression of MYB49 in tomato significantly enhanced the resistance of tomato to P. infestans, as evidenced by decreases in the number of necrotic cells, sizes of lesion, abundance of P. infestans, and disease index. Likewise, MYB49-overexpressing transgenic tomato plants also displayed increased tolerance to drought and salt stresses. Compared to WT plants, the accumulation of reactive oxygen species (ROS), malonaldehyde content, and relative electrolyte leakage was decreased, and peroxidase activity, superoxide dismutase activity, chlorophyll content, and photosynthetic rate were increased in MYB49-overexpressing tomato plants under P. infestans, salt or drought stress. These results suggested that tomato MYB49, as a positive regulator, could enhance the capacity to scavenge ROS, inhibit cell membrane damage and cell death, and protect chloroplasts, resulting in an improvement in resistance to P. infestans and tolerance to salt and drought stresses, and they provide a candidate gene for tomato breeding to enhance biotic stress resistance and abiotic stress tolerance.

Sly-miR1916 targets multiple target genes and negatively regulates the immune response in tomato: sly-miR1916 regulation of disease susceptibility

MicroRNA1916 (miR1916) is one of the nonconserved miRNAs that respond to various stresses in plants, but little has been known at present about its mechanisms in biotic stresses. In this study, the expression of Solanum lycopersicum (sly)-miR1916 in tomato was found to be down-regulated after infection with Phytophthora infestans or Botrytis cinerea. Tomato plants that overexpressed sly-miR1916 displayed significant enhancement in susceptibility to P.xa0infestans and B.xa0cinerea infection, as well as increased tendency to produce reactive oxygen species. Silencing of sly-miR1916 by short tandem target mimic and artificial microRNA strategies caused the tomato plants to become more tolerant to adverse conditions. In addition, lower sly-miR1916 expression could up-regulate the expression of strictosidine synthase (STR-2), UDP-glycosyltransferases (UGTs), late blight resistance protein homolog R1B-16, disease resistance protein RPP13-like, and MYB transcription factor (MYB12), which ultimately resulted in the accumulation of α-tomatine and anthocyanins via STR-2, UGT, and MYB12. Furthermore, ectopic expression of sly-miR1916/STR-2 significantly changed the tolerance of tobacco to B.xa0cinerea. Taken together, the results demonstrated that sly-miR1916 might regulate the expression of STR-2, UGT, and MYB12 in tomato plant, conferring sensitivity to biotic stress via modulating α-tomatine and anthocyanins.

Comparative transcriptome analysis shows the defense response networks regulated by miR482b

Key messageThe transcriptomic profile in the leaves of miR482b-overexpressing tomato plants revealed that miR482b may suppress alpha-linolenic acid metabolism, cysteine and methionine metabolism, plant–pathogen interaction, and the MAPK pathway to reduce resistance to Phytophthora infestans.AbstractOur previous study showed that tomato miR482b acted as a negative regulator during tomato resistance to Phytophthora infestans by silencing NBS-LRR genes. To investigate pathways related to miR482b, the transcriptomic profile of tomato plants that overexpressed miR482b was constructed. A total of 47,124,670 raw sequence reads from the leaves of miR482b-overexpressing tomato plants were generated by Illumina sequencing. A total of 746 genes in miR482b-overexpressing tomato plants were found to show significantly differential expression relative to those in wild-type tomato plants, including 132 up-regulated genes and 614 down-regulated genes. GO and KEGG enrichment analyses showed that plant–pathogen interaction, the MAPK pathway, and the pathways related to JA and ET biosynthesis were affected by miR482b in tomato. qRT-PCR results showed that all the enriched genes in these pathways were down-regulated in tomato plants that overexpressed miR482b and up-regulated in tomato plants that overexpressed an NBS-LRR gene (Soly02g036270.2, the target gene of miR482b). After P. infestans infection, the expression of the enriched genes showed a time-dependent response, and the genes played different roles between resistant tomato (Solanum pimpinellifolium L3708) and tomato susceptible to P. infestans (S. lycopersicum Zaofen No. 2). Our results have, therefore, demonstrated that miR482b is an important component of defense response network. This will also help to identify candidate genes involved in plant–pathogen interaction.