Xiuling Chen

Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28

Bacillus amyloliquefaciens TF28, an endophytic bacterium isolated from soybean root, showed strong antifungal activity in vitro. In this study, crude lipopeptides were extracted with methanol from the precipitate by adding concentrated HCl to culture filtrate. They exhibited highest antifungal activity against the rice bakanae fungus Fusarium moniliforme. Besides F. moniliforme, the crude lipopeptides also inhibited the growth of other phytopathogens such as Botrytis cinerea, Fusarium oxysporum etc. Microscopic analysis found that the crude lipopetides distorted hyphae and spore of F. moniliforme. The crude lipopetides were very stable to heat and insensitive to pH. They still retained strong antifungal activity after treatment at pH values ranging from 2 to 12 for 24 h or at 100°C for 30 min. Therefore, it is a candidate biocontrol agent for rice bakanae controlling. Biologically active fractions were isolated by high-performance liquid chromatography (HPLC). A component of a molecular weight of 1057 Da was identified as iturin A after electrospray ionization quadrupole time of flight tandem mass spectrometry analysis (ESI-Q-TOF-MS/MS).

Analysis of Clonostachys rosea-Induced Resistance to Tomato Gray Mold Disease in Tomato Leaves

Tomato gray mold disease, caused by Botrytis cinerea, is a serious disease in tomato. Clonostachys rosea is an antagonistic microorganism to B. cinerea. To investigate the induced resistance mechanism of C. rosea, we examined the effects of these microorganisms on tomato leaves, along with changes in the activities of three defense enzymes (PAL, PPO, GST), second messengers (NO, H2O2, O2 −) and phytohormones (IAA, ABA, GA3, ZT, MeJA, SA and C2H4). Compared to the control, all treatments induced higher levels of PAL, PPO and GST activity in tomato leaves and increased NO, SA and GA3 levels. The expression of WRKY and MAPK, two important transcription factors in plant disease resistance, was upregulated in C. rosea- and C. rosea plus B. cinerea-treated samples. Two-dimensional gel electrophoresis analysis showed that two abundant proteins were present in the C. rosea plus B. cinerea-treated samples but not in the other samples. These proteins were determined (by mass spectrum analysis) to be LEXYL2 (β-xylosidase) and ATP synthase CF1 alpha subunit. Therefore, C. rosea plus B. cinerea treatment induces gray mold resistance in tomato. This study provides a basis for elucidating the mechanism of C. rosea as a biocontrol agent.

A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members’ expression in tomato

Homeodomain-leucine zipper (HD-Zip) proteins are a kind of transcriptional factors that play a vital role in plant growth and development. However, no detailed information of HD-Zip family in tomato has been reported till now. In this study, 51 HD-Zip genes (SlHZ01-51) in this family were identified and categorized into 4 classes by exon–intron and protein structure in tomato (Solanum lycopersicum) genome. The synthetical phylogenetic tree of tomato, Arabidopsis and rice HD-Zip genes were established for an insight into their evolutionary relationships and putative functions. The results showed that the contribution of segmental duplication was larger than that of tandem duplication for expansion and evolution of genes in this family of tomato. The expression profile results under abiotic stress suggested that all SlHZ I genes were responsive to cold stress. This study will provide a clue for the further investigation of functional identification and the role of tomato HD-Zip I subfamily in plant cold stress responses and developmental events. Graphical Abstract Tomato HD-Zip family genes were classified into four groups by their gene structures and the highly homology with Arabidopsis and rice HD-Zip subfamily proteins.

Mapping and candidate gene screening of tomato Cladosporium fulvum-resistant gene Cf-19, based on high-throughput sequencing technology.

BackgroundTomato leaf mold is a common disease in tomato cultivation. This disease is caused by Cladosporium fulvum, which has many physiological races and differentiates rapidly. Cf genes confer resistance to C. fulvum, and the C. fulvum-tomato pathosystem is a model for the study of gene-for-gene interactions. Plants carrying the Cf-19 gene show effective resistance to C. fulvum in the field, and can be used in breeding and resistance mechanism studies as new resistant materials. In this study, we used F2 bulk specific-locus amplified fragment sequencing (SLAF-seq) and parental resequencing methods to locate and characterize the Cf-19 gene.ResultsA total of 4108 Diff_markers and three association regions were found in association analysis. A 2.14-Mb region containing seven Cf-type genes was identified in further analysis based on data from SLAF-seq and parental resequencing. Two candidate genes, Solyc01g006550.2.1 and Solyc01g005870.1.1, were screened out by quantitative real-time PCR (qRT-PCR) analysis. Sequence analysis showed that Solyc01g006550.2.1 (an allelic locus of Cf-0) in CGN18423 was a novel homologue of the Cladosporium resistance gene Cf-9 (Hcr9s) in the Cf-4/9 locus. The marker P7, which cosegregated with the resistant trait, was developed based on sequence mutation of the Solyc01g006550.2.1 locus in CGN18423.ConclusionsThe Cf-19 gene was mapped to the short arm of chromosome 1. The candidate genes Solyc01g006550.2.1 and Solyc01g005870.1.1 showed related amino acid sequence structures and expression patterns. Solyc01g006550.2.1 had a close evolutionary relationship with the functional Hcr9 members Cf-4 and Cf-9, and was very different from non-functional members. The results from this study will facilitate the breeding of cultivars carrying the Cf-19 gene and provide a basis for further gene cloning, resistance gene evolution and plant resistance mechanism studies.

Plastid DNA insertions in plant nuclear genomes: the sites, abundance and ages, and a predicted promoter analysis

The transfer of plastid DNA sequences into plant nuclear genomes plays an important role in the genomic evolution of plants. The abundance of nuclear-localized plastid DNA (nupDNA) correlates positively with nuclear genome size, but the genetic content of nupDNA remains unknown. In this mini review, we analyzed the number of nuclear-localized plastid gene fragments in known plant genomic data. Our analysis suggests that nupDNAs are abundant in plant nuclear genomes and can include multiple complete copies of protein-coding plastid genes. Mutated nuclear copies of plastid genes contained synonymous and nonsynonymous substitutions. We estimated the age of the nupDNAs based on the time when each integration occurred, which was calculated by comparing the nucleotide substitution rates of the nupDNAs and their respective plastid genes. These data suggest that there are two distinct age distribution patterns for nupDNAs in plants, and Oryza sativa and Zea mays were found to contain a very high proportion of young nupDNAs. Expressed sequence tags and predicted promoters of nupDNAs were identified, revealing that certain nuclear-localized plastid genes may be functional and that some have undergone positive natural selection pressure.

Transcriptome Analysis of the Sm-Mediated Hypersensitive Response to Stemphylium lycopersici in Tomato

Gray leaf spot disease caused by Stemphylium lycopersici is a major disease in cultivated tomato plants and threatens tomato-growing areas worldwide. Sm is a single dominant gene that confers resistance to tomato gray leaf spot disease agent. However, the underlying molecular mechanism remains unclear. Here, resistant (cv. Motelle, containing the Sm gene) and susceptible (cv. Moneymaker) plants were inoculated with virulent Stemphylium lycopersici isolate at a time point at which both cultivars showed a strong response to S. lycopersici infection. Transcriptome analyses were performed in both cultivars using RNA-seq. The number of differentially expressed genes (DEGs) was higher in Motelle than Moneymaker. Functional classification revealed that most DEGs were involved in plant–pathogen interactions, plant hormone signal transduction, regulation of autophagy, glycerophospholipid metabolism, and α-linolenic acid metabolism. Moreover, the genes that were significantly up-regulated in Sm tomatoes were involved in plant–pathogen interaction pathways. A total of 26 genes were selected for confirmation of differentially expressed levels by quantitative real-time PCR. This knowledge will yield new insights into the molecular mechanism of Sm responses to S. lycopersici infection.

Transcriptome Analysis of the Cf-12-Mediated Resistance Response to Cladosporium fulvum in Tomato

Cf-12 is an effective gene for resisting tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum). Unlike many other Cf genes such as Cf-2, Cf-4, Cf-5, and Cf-9, no physiological races of C. fulvum that are virulent to Cf-12 carrying plant lines have been identified. In order to better understand the molecular mechanism of Cf-12 gene resistance response, RNA-Seq was used to analyze the transcriptome changes at three different stages of C. fulvum infection (0, 4, and 8 days post infection [dpi]). A total of 9100 differentially expressed genes (DEGs) between 4 and 0 dpi, 8643 DEGs between 8 and 0 dpi and 2547 DEGs between 8 and 4 dpi were identified. In addition, we found that 736 DEGs shared among the above three groups, suggesting the presence of a common core of DEGs in response to C. fulvum infection. These DEGs were significantly enriched in defense-signaling pathways such as the calcium dependent protein kinases pathway and the jasmonic acid signaling pathway. Additionally, we found that many transcription factor genes were among the DEGs, indicating that transcription factors play an important role in C. fulvum defense response. Our study provides new insight on the molecular mechanism of Cf resistance to C. fulvum, especially the unique features of Cf-12 in responding to C. fulvum infection.

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

Key messageBased 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.AbstractLeaf 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.

The Endochitinase of Clonostachysrosea Expression in Bacillus amyloliquefaciens Enhances the Botrytis cinerea Resistance of Tomato

To investigate whether the ech42 gene in Clonostachysrosea can improve the biocontrol efficacy of Bacillus amyloliquefaciens and its molecular mechanism. Compared to the wild type, the B. amyloliquefaciens transformed with the ech42 gene exhibited higher chitinase activity. The B. amyloliquefaciens-ech42 also showed significantly higher biocontrol efficiency compared to Botrytiscinerea when tomato plants were pre-treated with B. amyloliquefaciens-ech42. No significant difference in biocontrol efficiency was observed between the wild type and B.amyloliquefaciens-ech42 when tomato plants were first infected by Botrytiscinerea. In addition, the activity of the defense-related enzyme polyphenol oxidase, but not superoxide dismutase, was significantly higher in B. amyloliquefaciens-ech42 than in the wild type. The ech42 enhances the biocontrol efficiency of B.amyloliquefaciens by increasing the capacity of preventative/curative effects in plants, rather than by killing the pathogens.