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A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers.

ABSTRACT: Fresh-cut roses (Rosa hybrida) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial B. cinerea inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose genes involved in B. cinerea resistance could provide useful genetic and genomic resources, we devised a virus-induced gene silencing (VIGS) procedure for the functional analysis of B. cinerea resistance genes in rose petals. We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar 'Samantha' showed the greatest decrease in RhPR10.1 expression among the cultivars tested. To determine whether jasmonic acid and ethylene are required for B. cinerea resistance in rose petals, we used VIGS to silence the expression of RhLOX5 and RhEIN3 (encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein, respectively) and found that petal susceptibility to B. cinerea was affected. Finally, a VIGS screen of B. cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B. cinerea resistance. Collectively, our data show that the combination of the DPDA and VIGS is a reliable and high-throughput method for studying B. cinerea resistance in rose.

SUBMITTER: Cao X

PROVIDER: S-EPMC6885046 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers.

Cao Xiaoqian X Yan Huijun H Liu Xintong X Li Dandan D Sui Mengjie M Wu Jie J Yu Hongqiang H Zhang Zhao Z

Horticulture research 20191201

Fresh-cut roses (Rosa hybrida) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial B. cinerea inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose g ...[more]

PMID: 31814989

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Project description:BackgroundRose is an important economic crop in horticulture. However, its field growth and postharvest quality are negatively affected by grey mould disease caused by Botrytis c. However, it is unclear how rose plants defend themselves against this fungal pathogen. Here, we used transcriptomic, metabolomic and VIGS analyses to explore the mechanism of resistance to Botrytis c.ResultIn this study, a protein activity analysis revealed a significant increase in defence enzyme activities in infected plants. RNA-Seq of plants infected for 0 h, 36 h, 60 h and 72 h produced a total of 54 GB of clean reads. Among these reads, 3990, 5995 and 8683 differentially expressed genes (DEGs) were found in CK vs. T36, CK vs. T60 and CK vs. T72, respectively. Gene annotation and cluster analysis of the DEGs revealed a variety of defence responses to Botrytis c. infection, including resistance (R) proteins, MAPK cascade reactions, plant hormone signal transduction pathways, plant-pathogen interaction pathways, Ca2+ and disease resistance-related genes. qPCR verification showed the reliability of the transcriptome data. The PTRV2-RcTGA1-infected plant material showed improved susceptibility of rose to Botrytis c. A total of 635 metabolites were detected in all samples, which could be divided into 29 groups. Metabonomic data showed that a total of 59, 78 and 74 DEMs were obtained for T36, T60 and T72 (T36: Botrytis c. inoculated rose flowers at 36 h; T60: Botrytis c. inoculated rose flowers at 60 h; T72: Botrytis c. inoculated rose flowers at 72 h) compared to CK, respectively. A variety of secondary metabolites are related to biological disease resistance, including tannins, amino acids and derivatives, and alkaloids, among others; they were significantly increased and enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways. This study provides a theoretical basis for breeding new cultivars that are resistant to Botrytis c.ConclusionFifty-four GB of clean reads were generated through RNA-Seq. R proteins, ROS signalling, Ca2+ signalling, MAPK signalling, and SA signalling were activated in the Old Blush response to Botrytis c. RcTGA1 positively regulates rose resistance to Botrytis c. A total of 635 metabolites were detected in all samples. DEMs were enriched in phenylpropanoid biosynthesis, glucosinolates and other disease resistance pathways.

Project description:For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the beta-tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446-bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of L-arginine, inadvertent gene silencing of bcass1 may result in partial L-arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild-type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or beta-glucuronidase using the pLOB1-based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.

Dataset Information

A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers.

Publications

A detached petal disc assay and virus-induced gene silencing facilitate the study of <i>Botrytis cinerea</i> resistance in rose flowers.

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