Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis

WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF is not well characterized. In the present study, we have characterized an OscWRKY1 gene from O. sanctum which shows induced expression after methyl jasmonate (MeJA), salicylic acid (SA), and wounding. Recombinant OscWRKY1 protein binds to the W-box cis-element TTGAC[C/T] and activates the reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have identified the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes and enhances the resistance against bacterial pathogen in Arabidopsis.

SlWRKY45 Interacts with Jasmonate-ZIM Domain Proteins to Negatively Regulate Defense Against Root Knot Nematode Meloidogyne incognita in Tomato

Root knot nematode (RKN), a kind of plant parasitic nematodes, leads to large reduction of crop yield, and seriously damages the agricultural production. The phytohormone jasmonates (JAs) act as important signals to regulate resistance against multiple abiotic and biotic stresses. However, little is known about the mechanism of JA-mediated defense responses against RKN in tomato. In this study, we found that the WRKY transcription factor SlWRKY45 interacts with most of the Jasmonate-ZIM domain proteins (JAZs) in yeast and plant. Overexpression of SlWRKY45 decreased plant resistance to RKN Meloidogyne incognita with increased gall index. We further generated slwrky45 mutants using the CRISPR/Cas9 technology, and discovered that the gall index and the number of nematodes and females in slwrky45 mutants are significantly reduced compared with wild type, as inoculated with RKN Meloidogyne incognita. Moreover, the contents of jasmonic acid and JA-isoleucine (JA-Ile) were highly increased in slwrky45 mutants with RKN Meloidogyne incognita infection compared with wild type. Furthermore, EMSA, and Dual-LUC assays demonstrated that SlWRKY45 directly binds and represses jasmonate biosynthesis gene ALLENE OXIDE CYCLASE ( AOC). Overall, our findings reveled that JAZ-interaction protein SlWRKY45 negatively controls plant defense against RKN Meloidogyne incognita by the regulation of JA biosynthesis in tomato.

Strawberry WRKY Transcription Factor WRKY50 Is Required for Resistance to Necrotrophic Fungal Pathogen Botrytis cinerea

WRKY protein is one of the largest plant-specific transcription factors that plays critical roles in plant stress responses, but few WRKY transcription factors have been functionally analyzed in strawberry. In this study, a Botrytis cinerea response WRKY gene, FvWRKY50, was isolated from the woodland strawberry. Expression analysis indicated that the transcript of FvWRKY50 was gradually decreased with fruit ripening, but was significantly induced by B. cinerea infection in mature strawberry fruit. Subcellular localization assay revealed that FvWRKY50 was localized in the nucleus. Several cis-elements related to pathogen responses were observed in the promoter region of FvWRKY50. Pathogen infection assay indicated that overexpression of FvWRKY50 in strawberry fruit significantly enhanced their resistance against B. cinerea, while the silencing of FvWRKY50 dramatically compromised their disease-resistant ability. The expression levels of several genes involved in jasmonic acid (JA) biosynthesis, signaling transduction, and antimicrobial protein biosynthesis were regulated to diverse extents in FvWRKY50 overexpressed and silenced fruit. Collectively, our study inferred that FvWRKY50 is a positive regulator that mediates resistance against B. cinerea through regulating some JA pathway and defense-related genes.

Exploring the Role of WRKY Transcription Factor in the Growth and Development of Bletilla striata based on Bioinformatics Analysis

WRKY type transcription factors (TFs) play crucial roles in the growth and development of plants. However, a comprehensive analysis of the WRKY family members in a valuable Chinese herbal orchid, Bletilla striata, or in other orchids, is limited. In this study, WRKY gene family was screened out from the transcriptome data of Bletilla striata by bioinformatics method. The 29 WRKY TFs that were identified from the B. striata genome and named BsWRKY1 to BsWRKY29 were divided into three clades: group Ⅰ (involving 8 WRKY sequences), group Ⅱ (18) and group Ⅲ (3), in which group Ⅱ was further divided into 5 subgroups: Ⅱ-a (involving 1 WRKY sequences), Ⅱ-b (5), and Ⅱ-c (3), Ⅱ-d (7), Ⅱ-e (3). EST-SSR marker mining test showed that 10 markers could be stably amplified with obvious polymorphisms among 4 landraces. Our data suggest that BsWRKY genes may work together to regulate plant growth and development. In different subcellular locations, BsWRKY genes not only played its own functions, but also coordinated the regulation of the whole life activities. Taken together, these results provided a theoretical basis for further studies on the gene functions and regulatory mechanisms of what in B. striata.

Applying HPLC to Screening QTLs for BLB Resistance in Rice

Bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae and is a major cause of rice yield reductions around the world. When diseased, plants produce a variety of metabolites to resist pathogens. In this study, the various defense metabolites were quantified using high-performance liquid chromatography (HPLC) after Xoo inoculation in a 120 Cheongcheong/Nagdong double haploid (CNDH) population. Quantitative trait locus (QTL) mapping was conducted using the concentration of the plant defense metabolites. HPLC analyzes the concentration of substances according to the severity of disease symptoms. Searching for BLB resistance candidate genes by applying this analysis method is very effective when mapping related genes. These resistance genes can be mapped directly to the causative pathogens. A total of 17 metabolites were detected by means of HPLC analysis after Xoo inoculation in the 120 CNDH population. QTL mapping of the metabolite concentrations resulted in the detection of the BLB resistance candidate gene, OsWRKYq6, in RM3343 of chromosome 6. OsWRKYq6 has a very high homology sequence with WRKY transcription factor 39, and when inoculated with Xoo, the relative expression level of the resistant population was higher than that of the susceptible population. Resistance genes have previously been detected using only phenotypic change data. In this study, resistance candidate genes were detected using the concentration of metabolites produced in plants after inoculation with pathogens. This newly developed analysis method can be used to effectively detect and identify genes directly involved in disease resistance for future studies.

GhWRKY1-like, a WRKY transcription factor, mediates drought tolerance in Arabidopsis via modulating ABA biosynthesis

Background Drought stress has great negative effects on the plant growth and development. The tolerance of plants to such abiotic stress is triggered by complicated and multilayered signaling pathways to restore cellular homeostasis and to promote survival. The WRKY family is one of the largest transcription factor families in higher plants, and has been well recognized for the roles in regulating plants tolerance to abiotic and biotic stress. However, little is known about how the WRKY genes regulate drought resistance in cotton. Results In this work, we identified the WRKY transcription factor GhWRKY1-like from upland cotton as a positive regulator of tolerance to drought that directly manipulates abscisic acid (ABA) biosynthesis. Overexpression of GhWRKY1-like in Arabidopsis constitutively activated ABA biosynthesis genes, signaling genes, responsive genes and drought related maker genes, and led to enhanced tolerance to drought. Further analysis has shown that GhWRKY1-like can interact with “W-box” cis-elements of the promoters of AtNCED2, AtNCED5, AtNCED6 and AtNCED9 which are essential enzymes for ABA biosynthesis, and promotes the expression of those target genes. Conclusions In summary, our findings suggest that GhWRKY1-like may act as a positive regulator in Arabidopsis tolerance to drought via directly interacting with the promoters of AtNCED2, AtNCED5, AtNCED6 and AtNCED9 to promote ABA biosynthesis.