VaBAM1 weakens cold tolerance by interacting with the negative regulator VaSR1 to suppress ß-amylase expression.

Cold stress is a key climatic factor that limits grape productivity and quality. Although ß-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1-overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1-overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1-overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance.

MicroRNA candidate miRcand137 in apple is induced by Botryosphaeria dothidea for impairing host defense.

MicroRNA (miRNA)-mediated gene silencing is a master gene regulatory pathway in plant-pathogen interactions. The differential accumulation of miRNAs among plant varieties alters the expression of target genes, affecting plant defense responses and causing resistance differences among varieties. Botryosphaeria dothidea is an important phytopathogenic fungus of apple (Malus domestica). Malus hupehensis (Pamp.) Rehder, a wild apple species, is highly resistant, whereas the apple cultivar "Fuji" is highly susceptible. Here, we identified a 22-nt miRNA candidate named miRcand137 that compromises host resistance to B. dothidea infection and whose processing was affected by precursor sequence variation between M. hupehensis and "Fuji." miRcand137 guides the direct cleavage of and produced target-derived secondary siRNA against Ethylene response factor 14 (ERF14), a transcriptional activator of pathogenesis-related homologs that confers disease resistance to apple. We showed that miRcand137 acts as an inhibitor of apple immunity by compromising ERF14-mediated anti-fungal defense and revealed a negative association between miRcand137 expression and B. dothidea sensitivity in both resistant and susceptible apples. Furthermore, MIRCAND137 was transcriptionally activated by the invading fungi but not by the fungal elicitor, implying B. dothidea induced host miRcand137 as an infection strategy. We propose that the inefficient miRcand137 processing in M. hupehensis decreased pathogen-initiated miRcand137 accumulation, leading to higher resistance against B. dothidea.

MdWRKY75e enhances resistance to Alternaria alternata in Malus domestica.

The Alternaria alternata apple pathotype adversely affects apple (Malus domestica Borkh.) cultivation. However, the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood. We have previously reported that MdWRKY75 expression is upregulated by A. alternata infection in 'Sushuai' apples. In this study, we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A. alternata infection, whereas silencing this gene enhanced susceptibility to A. alternata infection. Furthermore, we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A. alternata infection. Moreover, the thickening of the cell wall enhanced the mechanical defense capabilities of apple. In addition, we found that jasmonic acid remarkably induced MdWRKY75e expression, and its levels in transgenic apple lines were elevated. These results indicate that MdWRKY75e confers resistance to the A. alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants. In conclusion, our findings provide insights into the importance of MdWRKY75e for resistance to A. alternata infection in apples.

MicroRNA397b negatively regulates resistance of Malus hupehensis to Botryosphaeria dothidea by modulating MhLAC7 involved in lignin biosynthesis.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a vital role in the response of plants to pathogens. Although the microRNA397 family has been implicated in physiological processes as an important regulator, little is known about its function in the resistance of plants to pathogens. Here, Malus hupehensis miR397, which was induced by Botryosphaeria dothidea infection, was identified to directly target M. hupehensis Laccase7 (MhLAC7). The expression analysis of mature Mh-miR397 and MhLAC7 revealed their partly opposite expression patterns. The coexpression of Mh-miR397b in MhLAC7 overexpressing Nicotiana benthamiana suppressed the accumulation of exogenous MhLAC7 and endogenous NbLAC7, which led to decreased lignin content and reduced plant resistance to Botrytis cinerea. As reflected by increasing disease severity and pathogen growth, overexpression of miR397b in both the resistant M. hupehensis and susceptible M. domestica 'Gala' resulted in an increased sensitivity to B. dothidea infection, owing to reduced LAC7 expression and lignin content; however, the inhibition of miR397 had opposite effects. MicroRNA397 functions as a negative regulator in the resistance of Malus to B. dothidea by modulating the LAC7 expression and lignin biosynthesis.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

Selection of Suitable Reference Genes for RT-qPCR Normalization under Abiotic Stresses and Hormone Stimulation in Persimmon (Diospyros kaki Thunb).

The success of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to quantify gene expression depends on the stability of the reference genes used for data normalization. To date, systematic screening for reference genes in persimmon (Diospyros kaki Thunb) has never been reported. In this study, 13 candidate reference genes were cloned from 'Nantongxiaofangshi' using information available in the transcriptome database. Their expression stability was assessed by geNorm and NormFinder algorithms under abiotic stress and hormone stimulation. Our results showed that the most suitable reference genes across all samples were UBC and GAPDH, and not the commonly used persimmon reference gene ACT. In addition, UBC combined with RPII or TUA were found to be appropriate for the "abiotic stress" group and α-TUB combined with PP2A were found to be appropriate for the "hormone stimuli" group. For further validation, the transcript level of the DkDREB2C homologue under heat stress was studied with the selected genes (CYP, GAPDH, TUA, UBC, α-TUB, and EF1-α). The results suggested that it is necessary to choose appropriate reference genes according to the test materials or experimental conditions. Our study will be useful for future studies on gene expression in persimmon.

Genomic variants of genes associated with three horticultural traits in apple revealed by genome re-sequencing.

The apple (Malus × domestica Borkh.) cultivar 'Su Shuai' exhibits greater disease resistance, shorter internodes and lighter fruit flavor compared with its parents 'Golden Delicious' and 'Indo'. To obtain a comprehensive overview of the sequence variation in these three horticultural traits, the genomes of 'Su Shuai' and 'Indo' were resequenced using next-generation sequencing and compared to the genome of 'Golden Delicious'. A wide range of genetic variations were detected, including 2 454 406 and 18 749 349 single nucleotide polymorphism (SNP) and 59 547 and 50 143 structural variants (SVs) in the 'Indo' and 'Su Shuai' genomes, respectively. Among the SVs in 'Su Shuai', 17 genes related to disease resistance, 10 genes related to Gibberellin (GA) and 19 genes associated with fruit flavor were identified. The expression patterns of eight of the SV genes were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of this study illustrate the genomic variation in these cultivars and provide evidence for a genetic basis for the horticultural traits of disease resistance, short internodes and lighter flavor exhibited in these cultivars. These results provide a genetic basis for the phenotypic characteristics of 'Su Shuai' and, as such, these SVs could serve as gene-specific molecular markers in maker-assisted breeding of apples.