Salicylic Acid Signaling Pathway Research Articles

CsWRKY11 cooperates with CsNPR1 to regulate SA-triggered leaf de-greening and reactive oxygen species burst in cucumber

Salicylic acid (SA) is a multi-functional phytohormone, regulating diverse processes of plant growth and development, especially triggering plant immune responses and initiating leaf senescence. However, the early SA signaling events remain elusive in most plant species apart from Arabidopsis, and even less is known about the multi-facet mechanism underlying SA-regulated processes. Here, we report the identification of a novel regulatory module in cucumber, CsNPR1-CsWRKY11, which mediates the regulation of SA-promoted leaf senescence and ROS burst. Our analyses demonstrate that under SA treatment, CsNPR1 recruits CsWRKY11 to bind to the promoter of CsWRKY11 to activate its expression, thus amplifying the primary SA signal. Then, CsWRKY11 cooperates with CsNPR1 to directly regulate the expression of both chlorophyll degradation and ROS biosynthesis related genes, thereby inducing leaf de-greening and ROS burst. Our study provides a solid line of evidence that CsNPR1 and CsWRKY11 constitute a key module in SA signaling pathway in cucumber, and gains an insight into the interconnected regulation of SA-triggered processes.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae

BackgroundVerticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar “Zhongzhimian No. 2.”ResultsThe strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth.ConclusionsThis study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Analysis of Methylesterase Gene Family in Fragaria vesca Unveils Novel Insights into the Role of FvMES2 in Methyl Salicylate-Mediated Resistance against Strawberry Gray Mold.

Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

Biocontrol agents modulate phyllosphere microbiota interactions against pathogen Pseudomonas syringae

The pathogen Pseudomonas syringae, responsible for a variety of diseases, poses a considerable threat to global crop yields. Emerging biocontrol strategies employ antagonistic microorganisms, utilizing phyllosphere microecology and systemic resistance to combat this disease. However, the interactions between phyllosphere microbial dynamics and the activation of the plant defense system remain poorly understood. Here we show significant alterations in phyllosphere microbiota structure and plant gene expression following the application of biocontrol agents. We reveal enhanced collaboration and integration of Sphingomonas and Methylobacterium within the microbial co-occurrence network. Notably, Sphingomonas inhibits P. syringae by disrupting pathogen chemotaxis and virulence. Additionally, both Sphingomonas and Methylobacterium activate plant defenses by upregulating pathogenesis-related gene expression through abscisic acid, ethylene, jasmonate acid, and salicylic acid signaling pathways. Our results highlighted that biocontrol agents promote plant health, from reconstructing beneficial microbial consortia to enhancing plant immunity. The findings enrich our comprehension of the synergistic interplays between phyllosphere microbiota and plant immunity, offering potential enhancements in biocontrol efficacy for crop protection.

Control of Peach Leaf Curl with Foliar Applications of Plant Immunity Inducers and Insights in Elicitation of Defense Responses against Taphrina deformans.

Taphrina deformans is the causal agent of leaf curl, a serious peach disease which causes significant losses in peach production worldwide. Nowadays, in order to control plant diseases, it is necessary to adopt novel and low-cost alternatives to conventional chemical fungicides. These promising strategies are targeted at eliciting host defense mechanisms via priming the host through the consecutive application of plant immunity inducers prior to pathogen challenge. In this study, we investigated whether chitosan or yeast cell wall extracts could provide enhanced tolerance against leaf curl in two-season field trials. Furthermore, we addressed the possible molecular mechanisms involved beyond the priming of immune responses by monitoring the induction of key defense-related genes. The efficacy of spraying treatments against peach leaf curl with both inducers was significantly higher compared to the untreated control, showing efficacy in reducing disease severity of up to 62.6% and 73.9% for chitosan and yeast cell wall extracts, respectively. The application of chitosan in combination with copper hydroxide was more efficient in reducing disease incidence and severity, showing efficacy values in the range of 79.5-93.18%. Peach plantlets were also spray-treated with immunity inducers three times prior to leaf inoculation with T. deformans blastospores in their yeast phase. The relative expression levels of nine key defense and priming genes, including those encoding members of pathogenesis-related (PR) proteins and hub genes associated with hormone biosynthesis, were monitored by RT-qPCR across three days after inoculation (dai). The results indicate that pre-treatments with these plant immunity inducers activated the induction of genes involved in salicylic acid (SA) and jasmonate (JA) defense signaling pathways that may offer systemic resistance, coupled with the upregulation of genes conferring direct antimicrobial effects. Our experiments suggest that these two plant immunity inducers could constitute useful components towards the effective control of T. deformans in peach crops.

TaCAP1 interacts with TaLHCB1s and positively regulates wheat resistance against stripe rust.

Actin filaments and their associated actin-binding proteins (ABPs) play key roles in plant innate immune signaling. CAP1, or cyclase-associated protein 1, is an important regulatory factor of the actin cytoskeleton-associated signaling network, and was hypothesized here to be involved in resistance against wheat stripe rust because TaCAP1 expression was up-regulated in response to Puccinia striiformis f. sp. tritici (Pst). Down regulation of TaCAP1 expression led to decreased resistance against Pst, in contrast to increased resistance upon TaCAP1 overexpressing, as demonstrated by the changes of phenotypes and hyphal growth. We found increased expression of pathogenesis-responsive or relative related genes and disease grade changed in TaCAP1 overexpressing plants. Our results also showed TaCAP1-regulated host resistance to Pst by inducing the production and accumulation of reactive oxygen species (ROS) and mediating the salicylic acid signaling pathway. Additionally, TaCAP1 interacted with chlorophyll a/b -binding proteins TaLHCB1.3 and TaLHCB1.4, also known as light-harvesting chlorophyll-protein complex II subunit B (LHCB), which belong to the light-harvesting complex II proteins family. Silencing of two TaLHCB1 genes showed higher susceptibility to Pst, which reduced wheat resistance against Pst. Therefore, the data presented herein further illuminate our understanding that TaCAP1 interacts with TaLHCB1s, and functions as a positive regulator of wheat resistance against the stripe rust.

Indole-3 acetic acid negatively regulates rose black spot disease resistance through antagonizing the salicylic acid signaling pathway via jasmonic acid.

IAA cooperates with JA to inhibit SA and negatively regulates rose black spot disease resistance. Black spot disease caused by the fungus Marssonina rosae is the most prevalent and severe ailment in rose cultivation, leading to the appearance of black spots on leaves and eventual leaf fall, significantly impacting the utilization of roses in gardens. Salicylic acid (SA) and jasmonic acid (JA) are pivotal hormones that collaborate with indole-3 acetic acid (IAA) in regulating plant defense responses; however, the detailed mechanisms underlying the induction of black spot disease resistance by IAA, JA, and SA remain unclear. In this study, transcript analysis was conducted on resistant (R13-54) and susceptible (R12-26) lines following M. rosae infection. In addition, the impact of exogenous interference with IAA on SA- and JA-mediated disease resistance was examined. The continuous accumulation of JA, in synergy with IAA, inhibited activation of the SA signaling pathway in the early infection stage, thereby negatively regulating the induction of effective resistance to black spot disease. IAA administration alleviated the inhibition of SA on JA to negatively regulate the resistance of susceptible strains by further enhancing the synthesis and accumulation of JA. However, IAA did not contribute to the negative regulation of black spot resistance when high levels of JA were inhibited. Virus-induced gene silencing of RcTIFY10A, an inhibitor of the JA signaling pathway, further suggested that IAA upregulation led to a decrease in disease resistance, a phenomenon not observed when the JA signal was inhibited. Collectively, these findings indicate that the IAA-mediated negative regulation of black spot disease resistance relies on activation of the JA signaling pathway.

Screening for disease resistance and profiling the expression of defense-related genes contributing to resistance against bacterial blight (Xanthomonas oryzae pv. oryzae) in rice genotypes

Bacterial Bight profoundly affects global rice-growing regions. Breeding resistant cultivars is a reliable, effective, and eco-friendly way to control bacterial blight. The study was conducted during 2021–2022 to evaluate the resistance of rice genotypes to bacterial blight under glasshouse conditions. A total of 137 rice genotypes were screened for their resistance against two Xanthomonas oryzae pv. oryzae strains (TXO01 and TXO08) using the leaf clipping method. The results revealed diverse disease reactions among the genotypes, with some exhibiting high levels of resistance characterized by hypersensitive reactions and shorter lesion lengths. To further investigate the genetic basis of resistance, we screened moderately resistant and resistant genotypes for major bacterial blight resistance genes, including Xa21, xa5, and xa13, using gene-specific markers. Five genotypes viz., ADT55, CB MAS 13056, CB MAS 13060, CB MAS 13066, and ACM 18234 confirmed the presence of all R genes in a homozygous state, indicating a potentially robust resistance background. In addition, we examined the expression patterns of five defense-related genes involved in the salicylic acid (SA) and jasmonic acid (JA) signaling pathways using quantitative real-time PCR (qRT-PCR). The resistant genotypes exhibited significantly elevated expression levels of OsNPR1, OsWRKY45, OsPAL1, OsPR1a, and OsPR10a, ranging from 2 to 20-fold changes compared with the susceptible genotype. In conclusion, our findings suggest that the resistant genotypes not only harbor multiple bacterial blight resistance genes but also manifest up regulated defense responses against Xanthomonas oryzae pv. oryzae, possibly substantiating their elevated level of disease resistance.

Pattern-Triggered Immunity and Effector-Triggered Immunity: crosstalk and cooperation of PRR and NLR-mediated plant defense pathways during host-pathogen interactions.

The elucidation of the molecular basis underlying plant-pathogen interactions is imperative for the development of sustainable resistance strategies against pathogens. Plants employ a dual-layered immunological detection and response system wherein cell surface-localized Pattern Recognition Receptors (PRRs) and intracellular Nucleotide-Binding Leucine-Rich Repeat Receptors (NLRs) play pivotal roles in initiating downstream signalling cascades in response to pathogen-derived chemicals. Pattern-Triggered Immunity (PTI) is associated with PRRs and is activated by the recognition of conserved molecular structures, known as Pathogen-Associated Molecular Patterns. When PTI proves ineffective due to pathogenic effectors, Effector-Triggered Immunity (ETI) frequently confers resistance. In ETI, host plants utilize NLRs to detect pathogen effectors directly or indirectly, prompting a rapid and more robust defense response. Additionally epigenetic mechanisms are participating in plant immune memory. Recently developed technologies like CRISPR/Cas9 helps in exposing novel prospects in plant pathogen interactions. In this review we explore the fascinating crosstalk and cooperation between PRRs and NLRs. We discuss epigenomic processes and CRISPR/Cas9 regulating immune response in plants andrecent findings that shed light on the coordination of these defense layers. Furthermore, we alsohave discussed the intricate interactions between the salicylic acid and jasmonic acid signalling pathways in plants, offering insights into potential synergistic interactions that would be harnessed for the development of novel and sustainable resistance strategies against diverse group of pathogens.

Effect of Salicylic Acid Treatment on Disease Resistance to Coleosporium zanthoxyli in Chinese Pepper (Zanthoxylum armatum).

The fungus Coleosporium zanthoxyli causes leaf rust in Chinese pepper (Zanthoxylum armatum). To investigate the control effect of elicitor treatment on leaf rust in this species, the impact of salicylic acid (SA) on the spores and growth of C. zanthoxyli and the induced resistance to leaf rust by Z. armatum were analyzed, and the possible defense mechanisms involved in SA induction were evaluated. The results showed that SA had no effect on C. zanthoxyli spore germination and growth; however, rust resistance was induced in Z. armatum. The optimal SA treatment concentration was 0.4 mg/ml, and the relative cure effect reached 44.56%. SA-induced disease resistance was evident for up to 10 days, while the optimal induction interval was 48 h after stimulation. Consistent with the induced resistance, H2O2, total protein, total phenol, and lignin concentrations and polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), superoxide dismutase (SOD), and catalase (CAT) activities were significantly increased with the SA treatment, whereas the malondialdehyde content was significantly decreased. In addition, exogenous SA promoted defense-related enzyme activities, including those of POD, CAT, and PAL, and increased H2O2, lignin, and endogenous SA contents. Furthermore, SA induced the expression of SA signaling pathway genes such as ZaPR1 and ZaNPR1, and silencing ZaPR1 enhanced the sensitivity of Z. armatum to leaf rust. Our results demonstrated that 0.4 mg/ml SA priming increased the activities of CAT, POD, and PAL, elevated the contents of H2O2, lignin, and endogenous SA, and upregulated the expression of the SA-related gene ZaPR1, thereby enhancing the resistance of Z. armatum to leaf rust.

Methyltransferase TaSAMT1 mediates wheat freezing tolerance by integrating brassinosteroid and salicylic acid signaling.

Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1, that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops.

INDUCER OF CBF EXPRESSION 1 promotes cold-enhanced immunity by directly activating salicylic acid signaling.

Cold stress affects plant immune responses, and this process may involve the salicylic acid (SA) signaling pathway. However, the underlying mechanism by which low temperature signals coordinate with SA signaling to regulate plant immunity remains unclear. Here, we found that low temperatures enhanced the disease resistance of Arabidopsis thaliana against Pseudomonas syringae pv. tomato (Pst) DC3000. This process required INDUCER OF CBF EXPRESSION 1 (ICE1), the core transcription factor in cold-signal cascades. ICE1 physically interacted with NON-EXPRESSER OF PR GENES 1 (NPR1), the master regulator of the SA signaling pathway. Enrichment of ICE1 on the PATHOGENESIS-RELATED GENE 1 (PR1) promoter and its ability to transcriptionally activate PR1 were enhanced by NPR1. Further analyses revealed that cold stress signals cooperate with SA signals to facilitate plant immunity against pathogen attack in an ICE1-dependent manner. Cold treatment promoted interactions of NPR1 and TGACG-BINDING FACTOR 3 (TGA3) with ICE1, and increased the ability of the ICE1-TGA3 complex to transcriptionally activate PR1. Together, our results characterize a critical role of ICE1 as an indispensable regulatory node linking low temperature-activated and SA-regulated immunity. Understanding this crucial role of ICE1 in coordinating multiple signals associated with immunity broadens our understanding of plant-pathogen interactions.

The Role of Plant Defense Signaling Pathways in Phytoplasma-Infected and Uninfected Aster Leafhoppers' Oviposition, Development, and Settling Behavior.

In plant-microbe-insect systems, plant-mediated responses involve the regulation and interactions of plant defense signaling pathways of phytohormones jasmonic acid (JA), ethylene (ET), and salicylic acid (SA). Phytoplasma subgroup 16SrI is the causal agent of Aster Yellows (AY) disease and is primarily transmitted by populations of aster leafhoppers (Macrosteles quadrilineatus Forbes). Aster Yellows infection in plants is associated with the downregulation of the JA pathway and increased leafhopper oviposition. The extent to which the presence of intact phytohormone-mediated defensive pathways regulates aster leafhopper behavioral responses, such as oviposition or settling preferences, remains unknown. We conducted no-choice and two-choice bioassays using a selection of Arabidopsis thaliana lines that vary in their defense pathways and repeated the experiments using AY-infected aster leafhoppers to evaluate possible differences associated with phytoplasma infection. While nymphal development was similar among the different lines and groups of AY-uninfected and AY-infected insects, the number of offspring and individual female egg load of AY-uninfected and AY-infected insects differed in lines with mutated components of the JA and SA signaling pathways. In most cases, AY-uninfected insects preferred to settle on wild-type (WT) plants over mutant lines; no clear pattern was observed in the settling preference of AY-infected insects. These findings support previous observations in other plant pathosystems and suggest that plant signaling pathways and infection with a plant pathogen can affect insect behavioral responses in more than one manner. Potential differences with previous work on AY could be related to the specific subgroup of phytoplasma involved in each case.

NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

The Vitis yeshanensis U-box E3 ubiquitin ligase VyPUB21 enhances resistance to powdery mildew by targeting degradation of NIM1-interacting (NIMIN) protein.

VyPUB21 plays a key role during the defense against powdery mildew in grapes. Ubiquitin-ligating enzyme (E3), a type of protein widely found in plants, plays a key role in their resistance to disease. Yet how E3 participates in the disease-resistant response of Chinese wild grapevine (Vitis yeshanensis) remains unclear. Here we isolated and identified a U-box type E3 ubiquitin ligase, VyPUB21, from V. yeshanensis. This gene's expression level rose rapidly after induction by exogenous salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) and powdery mildew. In vitro ubiquitination assay results revealed VyPUB21 could produce ubiquitination bands after co-incubation with ubiquitin, ubiquitin-activating enzyme (E1), and ubiquitin-conjugating enzyme (E2); further, mutation of the conserved amino acid site in the U-box can inhibit the ubiquitination. Transgenic VyPUB21 Arabidopsis had low susceptibility to powdery mildew, and significantly fewer conidiophores and spores on its leaves. Expression levels of disease resistance-related genes were also augmented in transgenic Arabidopsis, and its SA concentration also significantly increased. VyPUB21 interacts with VyNIMIN and targets VyNIMIN protein hydrolysis through the 26S proteasome system. Thus, the repressive effect of the NIMIN-NPR complex on the late systemic acquired resistance (SAR) gene was attenuated, resulting in enhanced resistance to powdery mildew. These results indicate that VyPUB21 encoding ubiquitin ligase U-box E3 activates the SA signaling pathway, and VyPUB21 promotes the expression of late SAR gene by degrading the important protein VyNIMIN of SA signaling pathway, thus enhancing grape resistance to powdery mildew.

Constitutive Overexpression of an NB-ARC Gene from Wild Chinese Vitis quinquangularis in Arabidopsis thaliana Enhances Resistance to Phytopathogenic Oomycete and Bacteria.

Resistance (R) genes were used to recognize pathogen effectors directly or indirectly in plants and activate defense signal pathways. Most of these R proteins consist of a nucleotide-binding adaptor (NB-ARC) domain, a leucine-rich repeat (LRR) domain and some also have a coiled-coil (CC) structure. In this study, we cloned a gene which encodes the CC-NB-ARC-LRR R protein (VqCNL) from Chinese wild grapevine Vitis. quinquangularis accession 'Dan-2'. The transcript of VqCNL was obviously induced by inoculation with Plasmopara viticola and the salicylic acid (SA) treatment. The results of sequence analysis showed that the VqCNL gene contained a CC domain at the N-terminus, along with an NB-ARC and an LRR domain at the C-terminus. We transferred this gene into wildtype Arabidopsis and treated transgenic lines with Hyaloperonospora arabidopsidis (Hpa) and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000); the results demonstrated that VqCNL promotes broad spectrum resistance to pathogens. Furthermore, qPCR analysis displayed that VqCNL may display a significant function in disease resistance via activating SA signaling pathways. In general, these conclusions primarily demonstrated that VqCNL enhances the disease resistance level in plants and contributes to future research of the R gene identification for grape breeding biotechnology.

Genome-wide identification of GA2ox genes family and analysis of PbrGA2ox1-mediated enhanced chlorophyll accumulation by promoting chloroplast development in pear

BackgroundChlorophyll (Chl) is an agronomic trait associated with photosynthesis and yield. Gibberellin 2-oxidases (GA2oxs) have previously been shown to be involved in Chl accumulation. However, whether and how the PbrGA2ox proteins (PbrGA2oxs) mediate Chl accumulation in pear (Pyrus spp.) is scarce.ResultsHere, we aimed to elucidate the role of the pear GA2ox gene family in Chl accumulation and the related underlying mechanisms. We isolated 13 PbrGA2ox genes (PbrGA2oxs) from the pear database and identified PbrGA2ox1 as a potential regulator of Chl accumulation. We found that transiently overexpressing PbrGA2ox1 in chlorotic pear leaves led to Chl accumulation, and PbrGA2ox1 silencing in normal pear leaves led to Chl degradation, as evident by the regreening and chlorosis phenomenon, respectively. Meanwhile, PbrGA2ox1-overexpressing (OE) tobacco plants discernably exhibited Chl built-up, as evidenced by significantly higher Pn and Fv/Fm. In addition, RNA sequencing (RNA-seq), physiological and biochemical investigations revealed an increase in abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) concentrations and signaling pathways; a marked elevation in reducing and soluble sugar contents; and a marginal decline in the starch and sucrose levels in OE plants. Interestingly, PbrGA2ox1 overexpression did not prominently affect Chl synthesis. However, it indeed facilitated chloroplast development by increasing chloroplast number per cell and compacting the thylakoid granum stacks. These findings might jointly contribute to Chl accumulation in OE plants.ConclusionOverall, our results suggested that GA2oxs accelerate Chl accumulation by stimulating chloroplast development and proved the potential of PbrGA2ox1 as a candidate gene for genetically breeding biofortified pear plants with a higher yield.

The glycoside hydrolase 7 member VdGH7a regulates Verticillium dahliae pathogenicity and induces host defenses by interacting with GhOLP11

Pathogens secrete multiple enzymes that can degrade the cell wall, thereby weakening the host’s cell wall and facilitating the penetration of the pathogen into the plant. In this study, we identified VdGH7a, a glycoside hydrolase family 7 (GH7) cellobiohydrolase from Verticillium dahliae, which exhibited hydrolytic activity against 1,4-β-glucan. Interestingly, we found that VdGH7a induced cell death in Nicotiana benthamiana when signal peptides were present. However, this phenomenon was effectively prevented by the carbohydrate-binding type-1 (CBM1) protein domain. Furthermore, we observed that the knockout of VdGH7a significantly reduced the pathogenicity of V. dahliae to cotton plant, as evidenced by the inability of the knockout mutants to penetrate cellophane membrane. Additionally, these knockout mutants displayed diminished ability to exploit carbon sources, rendering them more susceptible to osmotic and cell wall stresses. Moreover, VdGH7a interacted with an osmotin-like protein (GhOLP1) in cotton through yeast two-hybrid screening, and further confirmed using bi-molecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI). Furthermore, virus-induced gene silencing technology was employed to silence GhOLP1, causing cotton’s salicylic acid (SA) content and resistance to V. dahliae were both reduced, whereas heterologous overexpression of GhOLP1 in Arabidopsis increased both resistance and the expression of genes involved in the SA signaling pathway. Collectively, these findings demonstrate a virulence strategy whereby the secreted protein VdGH7a from V. dahliae interacts with GhOLP1 to stimulate host immunity and play a significant role in plant resistance against V. dahliae.

Quinolizidine Alkaloids and Isoflavones from the Herb of Thermopsis lupinoides and Their Antiviral, Antifungal, and Insecticidal Activities.

As part of our ongoing investigation of natural bioactive substances from the genus Thermopsis of the tribe Fabaceae for agricultural protection, the chemical constituents of the herb Thermopsis lupinoides were systematically investigated, which led to the isolation of 39 quinolizidine alkaloids (QAs) (1-39), including 14 new QAs (1-14) and 14 isoflavones (40-53). Their structures were elucidated through comprehensive spectroscopic data analysis (IR, UV, NMR, HRESIMS), ECD calculations, and X-ray crystallography. The antitomato spotted wilt virus (TSWV) and antifungal (against Botrytis cinerea, Gibberella zeae, Phytophythora capsica, and Alternaria alternata) and insecticidal (against Aphis fabae and Tetranychus urticae) activities of the isolated compounds were screened using the lesion counting method, mycelial inhibition assay, and spray method, respectively. The bioassay results showed that 34 exhibited excellent protective activity against TSWV, with an EC50 value of 36.04 μg/mL, which was better than that of the positive control, ningnanmycin (86.03 μg/mL). The preliminary mechanistic exploration illustrated that 34 induced systemic acquired resistance in the host plant by acting on the salicylic acid signaling pathway. Moreover, 1 showed significant antifungal activity against B. cinerea (EC50 value of 20.83 μg/mL), while 2 exhibited good insecticidal activity against A. fabae (LC50 value of 24.97 μg/mL). This research is promising for the invention of novel pesticides from QAs with high efficiency and satisfactory ecological compatibility.

Comprehensive phytohormone metabolomic and transcriptomic analysis of tobacco (Nicotiana tabacum) infected by tomato spotted wilt virus (TSWV)

Tomato spotted wilt virus (TSWV) is ranked among the top 10 most destructive viruses globally. It results in abnormal leaf growth, stunting, and even death, significantly affecting crop yield and quality. Phytohormones play a crucial role in regulating plant-virus interactions. However, there is still limited research on the effect of TSWV on phytohormone levels, particularly growth hormones and genes involved in the phytohormone pathway. In our study, we combined phytohormone metabolomics and transcriptomics to examine the impact of TSWV infection on phytohormone content and gene expression profile. Metabolomic results showed that 41 metabolites, including major phytohormones and their precursors and derivatives were significantly altered after 14 days of TSWV inoculation tobacco plants cvK326, with 31 being significantly increased and 10 significantly reduced. Specifically, the levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) were significantly reduced. The levels of indole-3-acetic acid (IAA) have remained unchanged. However, the levels of cytokinin isopentenyladenine (iP) and salicylic acid (SA) significantly increased. The transcriptome analysis revealed 2,746 genes with significant changes in expression. Out of these, 1,072 genes were significantly downregulated, while 1,674 genes were significantly upregulated. Among them, genes involved in ABA synthesis and signaling pathways, such as 9-cis-epoxycarotenoid dioxygenase (NCED), protein phosphatase 2C (PP2C), serine/threonine-protein kinase (SnRK2), and abscisic acid responsive element binding factor (ABF), exhibited significant downregulation. Additionally, expression of the lipoxygenase gene LOX, Jasmonate ZIM domain-containing protein gene JAZ, and transcription factor gene MYC were significantly down-regulated. In the cytokinin pathway, while there were no significant changes in the expression of the cytokinin synthesis genes, a significant downregulation of transcriptionally active factor type-B response regulators (type-B RRs) was observed. In terms of SA synthesis and signaling pathways, the isochorismate synthase gene ICS1 and the pathogenesis-related gene PR1 were significantly upregulated. These results can strengthen the theoretical foundation for understanding the interaction between TSWV and tobacco and provide new insights for the future prevention and control of TSWV.