Aoxue Wang

Ectopic Overexpression of SlHsfA3, a Heat Stress Transcription Factor from Tomato, Confers Increased Thermotolerance and Salt Hypersensitivity in Germination in Transgenic Arabidopsis

Plant heat stress transcription factors (Hsfs) are the critical components involved in mediating responses to various environmental stressors. However, the detailed roles of many plant Hsfs are far from fully understood. In this study, an Hsf (SlHsfA3) was isolated from the cultivated tomato (Solanum lycopersicum, Sl) and functionally characterized at the genetic and developmental levels. The nucleus-localized SlHsfA3 was basally and ubiquitously expressed in different plant organs. The expression of SlHsfA3 was induced dramatically by heat stress, moderately by high salinity, and slightly by drought, but was not induced by abscisic acid (ABA). The ectopic overexpression of SlHsfA3 conferred increased thermotolerance and late flowering phenotype to transgenic Arabidopsis plants. Moreover, SlHsfA3 played a negative role in controlling seed germination under salt stress. RNA-sequencing data demonstrated that a number of heat shock proteins (Hsps) and stress-associated genes were induced in Arabidopsis plants overexpressing SlHsfA3. A gel shift experiment and transient expression assays in Nicotiana benthamiana leaves demonstrated that SlHsfA3 directly activates the expression of SlHsp26.1-P and SlHsp21.5-ER. Taken together, our results suggest that SlHsfA3 behaves as a typical Hsf to contribute to plant thermotolerance. The late flowering and seed germination phenotypes and the RNA-seq data derived from SlHsfA3 overexpression lines lend more credence to the hypothesis that plant Hsfs participate in diverse physiological and biochemical processes related to adverse conditions.

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.

Ectopic Overexpression of SsCBF1, a CRT/DRE-Binding Factor from the Nightshade Plant Solanum lycopersicoides, Confers Freezing and Salt Tolerance in Transgenic Arabidopsis

The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193–228 (SsCBF1193–228). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

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.

Isolation and functional characterization of the ShCBF1 gene encoding a CRT/DRE-binding factor from the wild tomato species Solanum habrochaites.

Plant growth and productivity are greatly affected by low ambient temperature. Complex cascades of gene expression in cold stress response are regulated by transcription factors. In this study, a cDNA clone, named ShCBF1, was isolated from Solanum habrochaites seedlings (a wild relative of cultivated tomato). It was classified as one of CBF family members based on multiple sequence alignment. The expression analysis confirmed that ShCBF1 was induced by low temperature, high salinity and drought stress. Experiments of subcellular localization in tobacco leaf cells indicated that it was localized in nucleus. Transient expression assay using onion epidermal cells revealed that the ShCBF1 protein could function similarly to AtCBF1 in activating the expression of reporter genes with a CRT/DRE element in their promoter. Moreover, ectopic overexpression of ShCBF1 in Arabidopsis enhanced freezing and high salinity tolerance of transgenic plants by improving the expression levels of some stress-responsive marker genes. Taken together, our results suggest that ShCBF1 behaves as a typical plant CBF transcription factor and might be involved in plant response to various environmental stresses.

Seed development and viviparous germination in one accession of a tomato rin mutant

In an experimental field, seed vivipary occurred in one accession of tomato rin mutant fruit at approximately 45–50 days after pollination (DAP). In this study, the possible contributory factors to this viviparous germination were investigated. Firstly, developing seeds were freshly excised from the fruit tissue every 5 days from 25–60 DAP. Germination occurred when isolated seeds were incubated on water, but was inhibited when they remained ex situ in fruit mucilage gel. The effect of abscisic acid (ABA) and osmoticum, separate and together, on germination of developing seeds was investigated. Additionally, ABA content in the seed and mucilage gel, as well as fruit osmolality were measured. The results showed that ABA concentrations in seeds were low during early development and increased later, peaking at about 50 DAP. ABA concentrations in rin accession were similar to those of the control cultivar and thus are not directly associated with the occurrence of vivipary. Developing seeds of rin accession are more sensitive than control seeds to all inhibitory compounds. However, osmolality in rin fruit at later developmental stages becomes less negative that is required to permit germination of developing seeds. Hence, hypo-osmolality in rin fruit may be an important factor in permitting limited viviparous germination.

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.

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.

Mapping and screening of the tomato Stemphylium lycopersici resistance gene, Sm , based on bulked segregant analysis in combination with genome resequencing

BackgroundTomato gray leaf spot disease caused by Stemphylium lycopersici (S. lycopersici) is considered one of the major diseases of cultivated tomatoes. The only S. lycopersici resistance gene, Sm, was derived from the wild tomato species S. pimpinellifolium. Sm has been identified as an effective source of gray leaf spot resistance in tomatoes and has been mapped to tomato chromosome 11. In this study, the first bulked segregant analysis (BSA) combined with genome resequencing for the mapping and screening of the Sm candidate gene was performed.ResultsBased on the resequencing results, we identified 50,968 Diff-markers, most of which were distributed on chromosome 11. A total of 37 genes were located in the interval of 0.26-Mb. The gene loci of resistant and susceptible lines were sequenced successfully using PCR products. The relative expression levels of candidate genes in resistant and susceptible lines were confirmed via qRT-PCR, Solyc11g011870.1.1 and Solyc11g011880.1.1 were identified through qRT-PCR. A marker, D5, which was cosegregated with the resistant locus, was identified according to the mutation of the Solyc11g011880.1.1 trait in the resistant line.ConclusionsThe Sm gene was mapped to the short arm of chromosome 11. The candidate genes Solyc11g011870.1.1 and Solyc11g011880.1.1 displayed expression patterns related to the resistance response. This study will be valuable for Sm cloning and Sm gene breeding in tomato.