Dataset Information

Transcriptome profiling of pumpkin (Cucurbita moschata Duch.) leaves infected with powdery mildew.

ABSTRACT: Cucurbit powdery mildew (PM) is one of the most severe fungal diseases, but the molecular mechanisms underlying PM resistance remain largely unknown, especially in pumpkin (Cucurbita moschata Duch.). The goal of this study was to identify gene expression differences in PM-treated plants (harvested at 24 h and 48 h after inoculation) and untreated (control) plants of inbred line "112-2" using RNA sequencing (RNA-Seq). The inbred line "112-2" has been purified over 8 consecutive generations of self-pollination and shows high resistance to PM. More than 7600 transcripts were examined in pumpkin leaves, and 3129 and 3080 differentially expressed genes (DEGs) were identified in inbred line "112-2" at 24 and 48 hours post inoculation (hpi), respectively. Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database and GO (Gene Ontology) database, a complex regulatory network for PM resistance that may involve hormone signal transduction pathways, transcription factors and defense responses was revealed at the transcription level. In addition, the expression profiles of 16 selected genes were analyzed using quantitative RT-PCR. Among these genes, the transcript levels of 6 DEGs, including bHLH87 (Basic Helix-loop-helix transcription factor), ERF014 (Ethylene response factor), WRKY21 (WRKY domain), HSF (heat stress transcription factor A), MLO3 (Mildew Locus O), and SGT1 (Suppressor of G-Two Allele of Skp1), in PM-resistant "112-2" were found to be significantly up- or down-regulated both before 9 hpi and at 24 hpi or 48 hpi; this behavior differed from that observed in the PM-susceptible material (cultivar "Jiujiangjiaoding"). The transcriptome data provide novel insights into the response of Cucurbita moschata to PM stress and are expected to be highly useful for dissecting PM defense mechanisms in this major vegetable and for improving pumpkin breeding with enhanced resistance to PM.

SUBMITTER: Guo WL

PROVIDER: S-EPMC5761878 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Transcriptome profiling of pumpkin (Cucurbita moschata Duch.) leaves infected with powdery mildew.

Guo Wei-Li WL Chen Bi-Hua BH Chen Xue-Jin XJ Guo Yan-Yan YY Yang He-Lian HL Li Xin-Zheng XZ Wang Guang-Yin GY

PloS one 20180110 1

Cucurbit powdery mildew (PM) is one of the most severe fungal diseases, but the molecular mechanisms underlying PM resistance remain largely unknown, especially in pumpkin (Cucurbita moschata Duch.). The goal of this study was to identify gene expression differences in PM-treated plants (harvested at 24 h and 48 h after inoculation) and untreated (control) plants of inbred line "112-2" using RNA sequencing (RNA-Seq). The inbred line "112-2" has been purified over 8 consecutive generations of sel ...[more]

PMID: 29320569

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Project description:Powdery mildew (PM), which is mainly caused by Podosphaera xanthii, is a serious biotrophic pathogen disease affecting field-grown and greenhouse-grown cucurbit crops worldwide. Because fungicides poorly control PM, the development and cultivation of PM-resistant varieties is critical. A homolog of SGT1 (suppressor of the G2 allele of skp1), which encodes a key component of the plant disease-associated signal transduction pathway, was previously identified through a transcriptomic analysis of a PM-resistant pumpkin (Cucurbita moschata) inbred line infected with PM. In this study, we have characterized this SGT1 homolog in C. moschata, and investigated its effects on biotic stress resistance. Subcellular localization results revealed that CmSGT1 is present in the nucleus. Additionally, CmSGT1 expression levels in the PM-resistant material was strongly induced by PM, salicylic acid (SA) and hydrogen peroxide (H2O2). In contrast, SA and H2O2 downregulated CmSGT1 expression in the PM-susceptible material. The ethephon (Eth) and methyl jasmonate (MeJA) treatments upregulated CmSGT1 expression in both plant materials. The constitutive overexpression of CmSGT1 in Nicotiana benthamiana (N. benthamiana) minimized the PM symptoms on the leaves of PM-infected seedlings, accelerated the onset of cell necrosis, and enhanced the accumulation of H2O2. Furthermore, the expression levels of PR1a and PR5, which are SA signaling transduction markers, were higher in the transgenic plants than in wild-type plants. Thus, the transgenic N. benthamiana plants were significantly more resistant to Erysiphe cichoracearum than the wild-type plants. This increased resistance was correlated with cell death, H2O2 accumulation, and upregulated expression of SA-dependent defense genes. However, the chlorosis and yellowing of plant materials and the concentration of bacteria at infection sites were greater in the transgenic N. benthamiana plants than in the wild-type plants in response to infections by the pathogens responsible for bacterial wilt and scab. Therefore, CmSGT1-overexpressing N. benthamiana plants were hypersensitive to these two diseases. The results of this study may represent valuable genetic information for the breeding of disease-resistant pumpkin varieties, and may also help to reveal the molecular mechanism underlying CmSGT1 functions.

Project description:BackgroundQuantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber-pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber-pumpkin grafted plants.MethodsIn this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber-pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder.ResultsThe results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber-pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber-pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting.DiscussionThis work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber-pumpkin grafted plants.

Dataset Information

Transcriptome profiling of pumpkin (Cucurbita moschata Duch.) leaves infected with powdery mildew.

Publications

Transcriptome profiling of pumpkin (Cucurbita moschata Duch.) leaves infected with powdery mildew.

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