Grapevine VaRPP13 protein enhances oomycetes resistance by activating SA signal pathway.

KEY MESSAGE: Expression of the VaRPP13 in Arabidopsis and tobacco enhanced resistance to oomycete pathogens, and this enhancement is closely related to the activation of salicylic acid (SA) signaling pathway. Resistance (R) genes, which usually contain a nucleotide-binding site and a leucine-rich repeat (NBS-LRR) domain, play crucial roles in disease resistance. In this study, we cloned a CC-NBS-LRR gene VaRPP13 from Vitis amurensis 'Shuang Hong' grapevine, and investigated its function on disease resistance. VaRPP13 expression was induced by Plasmopara viticola, an oomycetes pathogen causing downy mildew disease in grapevine. Heterologous expression VaRPP13 could also enhance resistance to Hyaloperonospora arabidopsidis in Arabidopsis thaliana and Phytophthora capsici in Nicotiana benthamiana, both oomycete pathogens. Further study indicated that VaRPP13 could enhance the expression of genes in SA signal pathway, while exogenous SA could also induce the expression of VaRPP13. In conclusion, our studies demonstrated that VaRPP13 contributes to a broad-spectrum resistance to oomycetes via activating SA signaling pathway.

Plant Defense Responses to a Novel Plant Elicitor Candidate LY5-24-2.

Plant elicitors enhance plant defense against pathogen attacks by inducing systemic acquired resistance (SAR) with no or low direct fungicidal activity. Here we report the synthesis of a novel plant elicitor candidate LY5-24-2 [3,4-dichloro-N-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)isothiazole-5-carboxamide] and evaluation of its SAR inducing activity. Bioassays indicated that LY5-24-2 did not show significant anti-fungal activity but provided long-lasting resistance in Arabidopsis thaliana (A. thaliana) through promoting the accumulation of lignin, cellulose and pectin by 60.1%, 82.4% and 305.6%, respectively, at a concentration of 100 µM. LY5-24-2 also facilitated the closure of leaf stomata and increased the intracellular free Ca2+ by 47.8%, induced reactive oxygen species (ROS) accumulation, and inhibited the activity of ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6) by 38.9% and 34.0%, respectively, as compared with the control at a concentration of 100 µM. LY5-24-2 induced SAR in plants and was dependent on the NPR1-mediated SA pathway by up-regulating expression of 2273 genes in A. thaliana. Meanwhile, LY5-24-2 also improved cucumber (Cucumis sativus L.) defense against Pseudoperonospora cubensis (P. cubensis) through promoting ROS accumulation and inhibiting activity of APX and CAT by 30.7% and 23.1%, respectively. Its expression of SA signaling genes CsNPR1, CsPR4 and CsPR5 was enhanced by 10.8, 5.8 and 6.6 times, respectively. These results demonstrated that LY5-24-2 is a novel elicitor candidate for plant protection via inducing SAR.

miR156/SPL9 Regulates Reactive Oxygen Species Accumulation and Immune Response in Arabidopsis thaliana.

The functions of microRNA156 (miR156) and its targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor genes in plant development have been widely investigated. However, the role of the miR156/SPLs regulatory network in plant immune systems remains obscure. Here, we found that the accumulation of reactive oxygen species (ROS) and the transcripts of basal salicylic acid (SA) signaling pathway genes were lower in Arabidopsis Pro35S:MIR156 seedlings (miR156 overexpression mutants) but higher in Pro35S:MIM156 (miR156 repression mutants) and ProSPL9:rSPL9 (SPL9 overexpression mutants) seedlings compared with wild-type Col-0 plants (WT). As a result, Pro35S:MIR156 mutants induced greater susceptibility to Pseudomonas syringae pv. tomato DC3000 following syringe infiltration than WT, while Pro35S:MIM156 and ProSPL9:rSPL9 mutants showed enhanced resistance. In addition, foliar H2O2 application resulted in activation of SA-mediated defense response and ablation of miR156-induced susceptibility to P. syringae pv. tomato DC3000 infection. Collectively, our results provide new insights into the function of the miR156/SPL network in Arabidopsis immune response by regulating ROS accumulation and activating the SA signaling pathway.

Identification and Characterization of High-Molecular-Weight Proteins Secreted by Plasmodiophora brassicae That Suppress Plant Immunity.

Plasmodiophora brassicae is an obligate intracellular parasitic protist that causes clubroot disease on cruciferous plants. So far, some low-molecular-weight secreted proteins from P. brassicae have been reported to play an important role in plant immunity regulation, but there are few reports on its high-molecular-weight secreted proteins. In this study, 35 putative high-molecular-weight secreted proteins (>300 amino acids) of P. brassicae (PbHMWSP) genes that are highly expressed during the infection stage were identified using transcriptome analysis and bioinformatics prediction. Then, the secretory activity of 30 putative PbHMWSPs was confirmed using the yeast signal sequence trap system. Furthermore, the genes encoding 24 PbHMWSPs were successfully cloned and their functions in plant immunity were studied. The results showed that ten PbHMWSPs could inhibit flg22-induced reactive oxygen burst, and ten PbHMWSPs significantly inhibited the expression of the SA signaling pathway marker gene PR1a. In addition, nine PbHMWSPs could inhibit the expression of a marker gene of the JA signaling pathway. Therefore, a total of 19 of the 24 tested PbHMWSPs played roles in suppressing the immune response of plants. Of these, it is worth noting that PbHMWSP34 can inhibit the expression of JA, ET, and several SA signaling pathway marker genes. The present study is the first to report the function of the high-molecular-weight secreted proteins of P. brassicae in plant immunity, which will enrich the theory of interaction mechanisms between the pathogens and plants.

Genome-wide analysis of MYB family in Nicotiana benthamiana and the functional role of the key members in resistance to Bemisia tabaci.

MYB transcription factors (TFs) play a key role in plant resistance to abiotic and biotical stresses. However, little is currently known about their involvement in the plant defense to piercing-sucking insects. Here, we studied the MYB TFs that responded to and resisted Bemisia tabaci whitefly in the model plant Nicotiana benthamiana. Firstly, a total of 453 NbMYB TFs in N. benthamiana genome were identified and 182 R2R3-MYB TFs were analyzed for molecular characteristics, phylogenetic analysis, genetic structure, motif composition, and cis-elements. Then, six stress-related NbMYB genes were selected for further study. The expression pattern shows they were highly expressed in mature leaves and intensively induced upon whitefly attack. Combined with bioinformatic analysis, overexpression, ß-Glucuronidase (GUS) assay, and virus-induced silencing tests, we determined the transcriptional regulation of these NbMYBs on the genes in lignin biosynthesis and SA-signaling pathways. Meanwhile, we tested the performance of whitefly on plants with increased or silenced NbMYB genes expression and found that NbMYB42, NbMYB107, NbMYB163, and NbMYB423 were resistant to whitefly. Our results contribute to a comprehensive understanding of the MYB TFs in N. benthamiana. Furthermore, our findings will facilitate further studies on the role of MYB TFs in the interaction between plants and piercing-sucking insects.

Overexpression of MdVQ37 reduces drought tolerance by altering leaf anatomy and SA homeostasis in transgenic apple.

Drought stress is an environmental factor that seriously threatens plant growth, development and yield. VQ proteins are transcriptional regulators that have been reported to be involved in plant growth, development and the responses to biotic and abiotic stressors. However, the relationship between VQ proteins and drought stress has not been well documented in plants. In this study, overexpressing the apple VQ motif-containing protein (MdVQ37) gene in apple plants markedly reduced the tolerance to drought. Physiological and biochemical studies further demonstrated lower enzymatic activities and decreased photosynthetic capacity in transgenic lines compared with wild-type (WT) plants under drought stress. Ultrastructural analysis of leaves showed that the leaves and palisade tissues from the transgenic lines were significantly thinner than those from WT plants. Salicylic acid (SA) analysis indicated that overexpression of MdVQ37 increased the accumulation of 2,5-DHBA by up-regulating the expression of the SA catabolic gene, which ultimately resulted to a significant reduction in endogenous SA content and the disruption of the SA-dependent signaling pathway under drought stress. Applying SA partially increased the survival rate of the transgenic lines under drought stress. These results demonstrate that the regulatory function of apple MdVQ37 is implicated in drought stress, through a change in leaf development and SA homeostasis. This study provides novel insight into understanding the multiple functions of VQ proteins.

Sugarcane responses to two strains of Xanthomonas albilineans differing in pathogenicity through a differential modulation of salicylic acid and reactive oxygen species.

Leaf scald caused by Xanthomonas albilineans is one of the major bacterial diseases of sugarcane that threaten the sugar industry worldwide. Pathogenic divergence among strains of X. albilineans and interactions with the sugarcane host remain largely unexplored. In this study, 40 strains of X. albilineans from China were distributed into three distinct evolutionary groups based on multilocus sequence analysis and simple sequence repeats loci markers. In pathogenicity assays, the 40 strains of X. albilineans from China were divided into three pathogenicity groups (low, medium, and high). Twenty-four hours post inoculation (hpi) of leaf scald susceptible variety GT58, leaf populations of X. albilineans strain XaCN51 (high pathogenicity group) determined by qPCR were 3-fold higher than those of strain XaCN24 (low pathogenicity group). Inoculated sugarcane plants modulated the reactive oxygen species (ROS) homoeostasis by enhancing respiratory burst oxidase homolog (ScRBOH) expression and superoxide dismutase (SOD) activity and by decreasing catalase (CAT) activity, especially after infection by X. albilineans XaCN51. Furthermore, at 24 hpi, plants infected with XaCN51 maintained a lower content of endogenous salicylic acid (SA) and a lower expression level of SA-mediated genes (ScNPR3, ScTGA4, ScPR1, and ScPR5) as compared to plants infected with XaCN24. Altogether, these data revealed that the ROS production-scavenging system and activation of the SA pathway were involved in the sugarcane defense response to an attack by X. albilineans.

Salicylic acid application alleviates cadmium accumulation in brown rice by modulating its shoot to grain translocation in rice.

Cadmium (Cd) contamination, which poses a serious threat to human health, has been recognized as a major threat to the agricultural system and crop production. Salicylic acid (SA) is a signaling molecule that plays an important role in against Cd toxicity. Previously, we found that spraying rice with SA could reduce the Cd accumulation in rice grains grown in Cd-contaminated soil. In this study, we studied the specific mechanism of SA spray on reducing Cd accumulation in rice grain. The results showed that treatment with SA could alleviate Cd toxicity in rice by increasing the activities of antioxidant enzymes that reduce hydrogen peroxide (H2O2) accumulation, but not by changing the pH, or total or available Cd of the soil. The key factor by which SA treatment reduced Cd accumulation in rice grains was by decreasing the Cd content in rice leaves at the flowering stage. This indicated that SA could modulate the Cd accumulation in shoots, reducing the Cd translocation to rice grains. Furthermore, SA could increase the H2O2 content, activating the SA-signaling pathway and modulating the expression levels of Cd transporters (OsLCT1 and OsLCD) in rice leaves to increase Cd tolerance and reduce Cd accumulation in the rice grain. Thus, spraying rice with SA may be effective measure to cope with Cd contamination in paddy soils.

AtWRKY1 negatively regulates the response of Arabidopsis thaliana to Pst. DC3000.

WRKY transcription factors (TFs) play a major role in resistance to plant diseases, but the role of AtWRKY1 in response to Pst. DC3000 is not clear. In this study, we found that AtWRKY1 negatively affected the response of Arabidopsis to Pst. DC3000. During Pst. DC3000 infection, the transcription of AtWRKY1 was suppressed. The wrky1 mutants displayed enhanced resistance to Pst. DC3000. In contrast, the overexpression of AtWRKY1 reduced the resistance. The relative RNA levels of defense related PR genes were increased in the loss-of-function mutants, whereas their expressions were decreased in the AtWRKY1-overexpressing plants. Further research revealed that salicylic acid (SA) can repress the expression of AtWRKY1, and overexpression of AtWRKY1 weakened the SA-mediated defense response. In addition, the AtWRKY1 protein can bind to the PR1 promoter in vivo and in yeast cells directly, thereby inhibiting the transcription of PR1. AtWRKY1 indirectly represses the expression of PR2 and PR5. Our results indicated that the AtWRKY1 gene negatively regulates the plant defense responses to Pst. DC3000 through SA signaling pathways.

Cotton CC-NBS-LRR Gene GbCNL130 Confers Resistance to Verticillium Wilt Across Different Species.

Verticillium wilt (VW) is a destructive disease in cotton caused by Verticillium dahliae and has a significant impact on yield and quality. In the absence of safe and effective chemical control, VW is difficult to manage. Thus, at present, developing resistant varieties is the most economical and effective method of controlling Verticillium wilt of cotton. The CC-NBS-LRR (CNL) gene family is an important class of plant genes involved in disease resistance. This study identified 141 GbCNLs in Gossypium barbadense genome, with 37.5% (53 genes) GbCNLs enriched in 12 gene clusters (GC01-GC12) based on gene distribution in the chromosomes. Especially, seven GbCNLs from two largest clusters (GC11 and GC12) were significantly upregulated in the resistant cultivar (Hai No. 7124) and the susceptible (Giza No. 57). Virus-induced gene silencing of GbCNL130 in G. barbadense, one typical gene in the gene cluster 12 (GC12), significantly altered the response to VW, compromising plant resistance to V. dahliae. In contrast, GbCNL130 overexpression significantly increased the resistance to VW in the wild-type Arabidopsis thaliana. Based on our research findings presented here, we conclude that GbCNL130 promotes resistance to VW by activating the salicylic acid (SA)-dependent defense response pathway resulting in strong accumulation of reactive oxygen species and upregulation of pathogenesis-related (PR) genes. In conclusion, our study resulted in the discovery of a new CNL resistance gene in cotton, GbCNL130, that confers resistance to VW across different hosts.

Isolation and functional identification of a Botrytis cinerea-responsive caffeoyl-CoA O-methyltransferase gene from Lilium regale wilson.

In plants, genes involved in the Phenylpropanoid/monolignol pathway play important roles in lignin biosynthesis and plant immunity. However, their biological function in Lilium remains poorly characterized. Comparative RNA sequencing of the expression profiles of the monolignol pathway genes from fungi-resistant species Lilium regale after inoculation with Botrytis cinerea was performed. One upregulated caffeoyl-CoA O-methyltransferase gene, LrCCoAOMT, was cloned for functional characterization by reverse genetic methods. LrCCoAOMT encodes a putative protein of 246 amino acids and is highly expressed in stem tissues and responsive to salicylic acid (SA) signaling and B. cinerea infection. LrCCoAOMT was largely directed to the cytoplasm. LrCCoAOMT overexpression in Arabidopsis resulted in an increased lignin deposition in vascular tissues and conferred resistance to B. cinerea infection in transgenic plants. Transient transformation of LrCCoAOMT in nonresistant Lilium sargentiae leaves also identified the defense function to B. cinerea. In addition, transcript levels of genes involved in the monolignol and SA-dependent signaling pathways were altered in transgenic Arabidopsis, suggesting that LrCCoAOMT might play vital roles in the resistance of L. regale to B. cinerea related to the levels of lignin and the regulation of SA signaling. This is the first report to functionally characterize a CCoAOMT gene in Lilium, a potential molecular target for lily molecular improvement.

Induction of resistance to diseases in plant by aerial ultrasound irradiation.

Ultrasound, which refers to frequencies above the audible limit of human hearing, is a candidate for inducing resistance to pathogens in plants. We revealed that aerial ultrasound of 40.5 kHz could induce disease resistance in tomatoes and rice when the plants were irradiated with ultrasound of ca. 100 dB for 2 weeks during nursery season and reduced the incidence of Fusarium wilt and blast diseases, respectively, when plants were inoculated with pathogen 0 or 1 week after terminating irradiation. Disease control efficacy was also observed with ultrasound at frequencies of 19.8 and 28.9 kHz. However, cabbage yellows and powdery mildew on lettuce were not suppressed by ultrasound irradiation. No significant positive or negative effect on growth was observed in tomato and rice plants. RT-qPCR showed that the expression of PR1a involved in the salicylic acid (SA) signaling pathway was upregulated in the ultrasound-irradiated tomato.