Endophyte-inoculated rhizomes of Paris polyphylla improve polyphyllin biosynthesis and yield: a transcriptomic analysis of the underlying mechanism.

Introduction: Polyphyllin from Paris polyphylla var. yunnanensis exhibits anti-inflammatory, analgesic, antibacterial, and antiviral properties. However, the current production of polyphyllin can barely meet market demand. To improve the content of polyphyllin produced by P. polyphylla, two endophyte strains, Bacillus cereus LgD2 and Fusarium oxysporum TPB, were isolated from Paris fargesii Franch. and inoculated in the roots of P. polyphylla. Both symbiotic strains significantly promoted the accumulation of saponins in P. polyphylla. Methods: The content of polyphyllin in rhizomes of P. polyphylla treated with TPB with LgD2 strain was determined using High Performance Liquid Chromatography and the expressed genes were analyzed by RNA-seq. Gene Ontology and Kyoto Encyclopedia of Genes annotations were performed on the differentially expressed genes, a clustering tree of UDP-glycosyltransferase (UGT) and cytochrome P450 (CYP450) gene families was constructed, and UGT and CYP450 involved in the biosynthesis of polyphyllin were predicted using weighted correlation network analysis (WGCNA). Results: RNA-seq and qRT-PCR analyses showed that endophytic inoculation did not promote polyphyllin accumulation by enhancing the upstream terpene biosynthesis pathway, but probably by up-regulating the downstream CYP450 and UGT genes associated with polyphyllin biosynthesis. Genomes enrichment analyses of differentially expressed genes indicated that inoculation with LgD2 and TPB played a positive role in promoting the defense against pathogenic bacteria, enhancing the biosynthesis of carbohydrates, attenuating the process of nitrogen metabolism, and maintaining the equilibrium of the redox reaction homeostasis, potentially indirectly enhancing the polyphyllin yield of P. polyphylla. By combining differentially expressed genes screening, WGCNA, and phylogenetic tree analyses, 17 CYP450 and 2 UGT candidate genes involved in the biosynthesis of polyphyllin I, polyphyllin II, polyphyllin VII, polyphyllin D, and polyphyllin H were identified. These results suggest that endophytes probably effectively promote the accumulation of polyphyllin by regulating key downstream genes in biosynthetic pathways. Discussion: This study provides a new approach for investigating the regulatory mechanisms of endophytes that promote the production and accumulation of polyphyllin in P. polyphylla, providing a basis for further elucidating the mechanisms of plant-endophyte interactions.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Limited Impact of Soil Microorganisms on the Endophytic Bacteria of Tartary Buckwheat (Fagopyrum tataricum).

Soil has been considered the main microbial reservoir for plants, but the robustness of the plant microbiome when the soil resource is removed has not been greatly considered. In the present study, we tested the robustness of the microbiota recruited by Tartary buckwheat (Fagopyrum tataricum Gaertn.), grown on sterile humus soil and irrigated with sterile water. Our results showed that the microbiomes of the leaf, stem, root and next-generation seeds were comparable between treated (grown in sterile soil) and control plants (grown in non-sterile soil), indicating that the plants had alternative robust ways to shape their microbiome. Seed microbiota contributed greatly to endophyte communities in the phyllosphere, rhizosphere and next-generation seeds. The microbiome originated from the seeds conferred clear benefits to seedling growth because seedling height and the number of leaves were significantly increased when grown in sterilized soil. The overall microbiome of the plant was affected very little by the removal of the soil microbial resource. The microbial co-occurrence network exhibited more interactions, and Proteobacteria was enriched in the root of Tartary buckwheat planted in sterilized soil. Our research broadens the understanding of the general principles governing microbiome assembly and is widely applicable to both microbiome modeling and sustainable agriculture.

AoSAP8-P encoding A20 and/or AN1 type zinc finger protein in asparagus officinalis L. Improving stress tolerance in transgenic Nicotiana sylvestris.

The full length CDS of an A20 and AN1 type zinc finger gene (named AoSAP8-P), located nearby the male specific Y chromosome (MSY) region of Asparagus officinalis (garden asparagus) was amplified by RT-PCR from purple passion. This gene, predicted as the stress associated protein (SAPs) gene families, encodes 172 amino acids with multiple cis elements including light, stress response box, MYB and ERF binding sites on its promoter. To analyze its function, the gene expression of different organs in different asparagus gender were analyzed and the overexpressed transgenic Nicotiana sylvestris lines were generated. The results showed the gene was highly expressed in both flower and root of male garden asparagus; the germination rate of seeds of the T2 transgenic lines (T2-5-4 and T2-7-1) under the stress conditions of 125 mM NaCl and 150 mM mannitol were significantly higher than the wild type (WT) respectively. When the potted T2-5-4, T2-7-1 lines and WT were subjected to drought stress for 24 days and the leaf discs immerged into 20 % PEG6000 and 300 mM NaCl solution for 48 h respectively, the T2-5-4 and T2-7-1 with AoSAP8-P expression showed stronger drought, salt and osmotic stress tolerance. When compared, the effects of AoSAP8-P overexpression on productive development showed that the flowering time of transgenic lines, were âˆ¼ 9 day earlier with larger but fewer pollens than its WT counterparts. However, there were no significant differences in anthers, stigmas and pollen viability between the transgenic lines and WT. Our results suggested that, the AoSAP8-P gene plays a role in improving the stress resistance and shortening seeds generation time for perianal survival during the growth and development of garden asparagus.

Cyclophilin effector Al106 of mirid bug Apolygus lucorum inhibits plant immunity and promotes insect feeding by targeting PUB33.

Apolygus lucorum (Meyer-Dur; Heteroptera: Miridae) is a major agricultural pest infesting crops, vegetables, and fruit trees. During feeding, A. lucorum secretes a plethora of effectors into its hosts to promote infestation. However, the molecular mechanisms of these effectors manipulating plant immunity are largely unknown. Here, we investigated the molecular mechanism underlying the effector Al106 manipulation of plant-insect interaction by RNA interference, electrical penetration graph, insect and pathogen bioassays, protein-protein interaction studies, and protein ubiquitination experiment. Expression of Al106 in Nicotiana benthamiana inhibits pathogen-associated molecular pattern-induced cell death and reactive oxygen species burst, and promotes insect feeding and plant pathogen infection. In addition, peptidyl-prolyl cis-trans isomerase (PPIase) activity of Al106 is required for its function to inhibit PTI.Al106 interacts with a plant U-box (PUB) protein, PUB33, from N. benthamiana and Arabidopsis thaliana. We also demonstrated that PUB33 is a positive regulator of plant immunity. Furthermore, an in vivo assay revealed that Al106 inhibits ubiquitination of NbPUB33 depending on PPIase activity. Our findings revealed that a novel cyclophilin effector may interact with plant PUB33 to suppress plant immunity and facilitate insect feeding in a PPIase activity-dependent manner.

Apolygus lucorum effector Al6 promotes insect feeding performance on soybean plants: RNAi analysis and feeding behaviour study with electrical penetration graph.

The mirid bug Apolygus lucorum, a dominant mirid species in northern China, is a notorious polyphagous pest with more than 200 hosts, including several major crops such as cotton and soybean, resulting in massive economic loss. Studies of insect salivary effectors may provide a novel control strategy for A. lucorum. An A. lucorum effector, that is, Al6, that inhibits plant immunity by using glutathione peroxidase to repress reactive oxidase accumulation was previously identified. In this study, we further explored the molecular functions of Al6 associated with feeding behaviour and insect survival on soybean, a major host of A. lucorum, using RNA interference and electrical penetration graph (EPG) techniques. We initially observed the injury symptom of this mirid bug and characterized feeding behaviour on soybean leaves using EPG. Our results revealed that A. lucorum preferred to feed on young plant organs such as tender leaves, shoots and buds. This mirid bug used cell rupture as a feeding strategy to ingest cell contents from plant tissues. Subsequently, we silenced the Al6 gene using RNAi and investigated the feeding behaviour, honeydew excretion, body weight, and survival rates of A. lucorum on soybean after Al6 knockdown. Our results demonstrated that silencing of Al6 significantly reduced feeding duration, amount of honeydew secretion, body weight, and survival rates of A. lucorum. Thus, our findings provide a novel molecular target of plant-mediated RNAi for the control of A. lucorum.

An updated network meta-analysis of EGFR-TKIs and combination therapy in the first-line treatment of advanced EGFR mutation positive non-small cell lung cancer.

Objectives: Tyrosine kinase inhibitors (TKIs) are a standard care option in patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutation. TKI-based combination treatment modes show encouraging outcomes. However, it remains unknown which is the optimal treatment as the first-line regimen for these patients on overall survival (OS). Materials and methods: Randomized controlled trials and meeting abstracts that investigated EGFR-TKIs alone or in combination as front-line care for patients with NSCLC were systematically searched in relevant databases and reviewed. Fixed and random effects network meta-analysis models were used to estimate progression-free survival (PFS), OS, overall response rate, and grade three and higher adverse events (AEs). Surface under the cumulative ranking curves (SUCRAs) were used to rank treatment effects. Results: Eighteen studies covering six treatments and involving a total of 4389 patients were included in this network meta-analysis. On OS, the top three treatment were first-generation EGFR-TKIs (1G EGFR-TKIs) plus chemotherapy (SUCRA, 88.1%), osimertinib (SUCRA, 65.8%) and second-generation EGFR-TKIs (2GEGFR-TKIs) (SUCRA, 63.3%). On PFS, the top three treatments were osimertinib (SUCRA, 96.0%), 1G EGFR-TKIs plus chemotherapy (SUCRA, 67.1%), and 1G EGFR-TKIs plus antiangiogenesis (SUCRA, 48.2%). Two types of TKI-based combination therapy have significantly higher risk of grade three and higher AEs than TKI alone. Conclusion: 1G EGFR-TKIs plus chemotherapy and osimertinib seem to be the two better options as first-line care in advanced NSCLC patients with EGFR-mutation. Osimertinib caused the lowest incidence of AEs. However, TKIs-based combination therapy significantly increased AEs.

Characterization of Salivary Secreted Proteins That Induce Cell Death From Riptortus pedestris (Fabricius) and Their Roles in Insect-Plant Interactions.

Riptortus pedestris (Fabricius) is a polyphagous hemipteran crop pest that mainly feeds on the leguminous plants, resulting in shriveled and dimpled seeds. With recent several outbreaks in the Huang-Huai-Hai region of China, as well as in South Korea and Japan, this species has caused enormous economic losses to soybean crops. In the present study, we found that R. pedestris feeding results in local lesions at the infestation sites. To identify the key effectors that induce plant damage during feeding, the salivary glands of R. pedestris were dissected for transcriptome sequencing, and 200 putative secreted proteins were transiently expressed in N. benthamiana. Among them, three intracellular effectors (RP191, RP246, and RP302) and one apoplastic effector (RP309) were identified as necrosis-inducing proteins (NIPs), which also triggered the reactive oxidative burst. Yeast signal sequence trap and qRT-PCR analysis suggested that these proteins might be secreted into plant tissue during R. pedestris infestation. Pathogenicity assays revealed that RP191, 246, and 302 promote Phytophthora capsici infection or induce Spodoptera litura feeding by inhibiting plant immunity. RP302 is localized to the cytoplasm and nuclei, while RP191 and 246 are endoplasmic reticulum (ER) resident proteins. RP309 stimulates the expression of PTI marker genes, and its induced cell death depends on co-receptors NbBAK1 and NbSOBIR1, indicating that it is a HAMP. Bioinformatics analysis demonstrated that four NIPs are recently evolved effectors and only conserved in the Pentatomidae. In this study, saliva-secreted proteins were used as the starting point to preliminarily analyze the harm mechanism of R. pedestris, which might provide a new idea and theoretical basis for this species control.

Feeding Behavior of Riptortus pedestris (Fabricius) on Soybean: Electrical Penetration Graph Analysis and Histological Investigations.

Riptortus pedestris (Fabricius) is a major agricultural pest feeding on soybean pods and seeds. The large populations occur during seed maturity stages from pod filling to harvest. Its infestation results in shriveled and dimpled seeds while vegetative structures (leaflet and stem) remain green, known as "Stay Green" syndrome. Additional evidence also demonstrates that soybean pods and seeds are required for Riptortus pedestris development. However, the feeding behavior strategies employed by this stink bug to feed on soybean plants are still not clear. In the present study, the feeding behaviors of R. pedestris on soybean plants were recorded by electropenetrography (EPG), and a waveform library was created for this species. A total of five phases of waveforms-nonprobing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4)-were identified. Non-probing waveforms Z and NP and pathway (Rp1) were found in all tested plant structures (leaflet, stem, cotyledon, and pods). Waveform Rp2 (xylem sap ingestion, xylem ingestion) was primarily recorded during R. pedestris feeding on leaflets and stems, while Rp3 (salivation/ingestion) was only observed during feeding on cotyledon and pods. Histological examinations confirmed that correlation between Rp2 and stylet tip positioning in the xylem vessel in leaflets and stems. Stylet tips end in the tissues of cotyledon and pods when Rp3 is recorded. Taken together, our results demonstrate that R. pedestris ingests xylem sap from vegetative tissues of soybean (leaflet and stem) via a salivary sheath strategy to obtain water. It mainly acquires nutrients from soybean pods and/or seeds using cell-rupture tactics. This study provided insightful information to understand the field occurrence patterns of "Stay Green" syndrome, which may have important implications for pest control.

Identification of Bioactive Components of Stephania epigaea Lo and Their Potential Therapeutic Targets by UPLC-MS/MS and Network Pharmacology.

Stephania epigaea, an important traditional folk medicinal plant, elucidating its bioactive compound profiles and their molecular mechanisms of action on human health, would better understand its traditional therapies and guide their use in preclinical and clinical. This study aims to detect the critical therapeutic compounds, predict their targets, and explore potential therapeutic molecular mechanisms. This work first determined metabolites from roots, stems, and flowering twigs of S. epigaea by a widely targeted metabolomic analysis assay. Then, the drug likeness of the compounds and their pharmacokinetic profiles were screened by the ADMETlab server. The target proteins of active compounds were further analyzed by PPI combing with GO and KEGG cluster enrichment analysis. Finally, the interaction networks between essential compounds, targets, and disease-associated pathways were constructed, and the essential compounds binding to their possible target proteins were verified by molecular docking. Five key target proteins (EGFR, HSP90AA1, SRC, TNF, and CASP3) and twelve correlated metabolites, including aknadinine, cephakicine, homostephanoline, and N-methylliriodendronine associated with medical applications of S. epigaea, were identified, and the compounds and protein interactions were verified. The key active ingredients are mainly accumulated in the root, which indicates that the root is the main medicinal tissue. This study demonstrated that S. epigaea might exert the desired disease efficacy mainly through twelve components interacting via five essential target proteins. EGFR is the most critical one, which deserves further verification by biological studies.

Age, male sex, higher posterior tibial slope, deep sulcus sign, bone bruises on the lateral femoral condyle, and concomitant medial meniscal tears are risk factors for lateral meniscal posterior root tears: a systematic review and meta-analysis.

PURPOSE: Lateral meniscus posterior root tears (LMPRTs) are commonly found in patients with anterior cruciate ligament (ACL) injuries. However, risk factors for LMPRTs are not well known. This study was designed to systematically review the available evidence regarding risk factors associated with LMPRTs. METHODS: The PubMed, EMBASE, Cochrane Library, and Web of Science databases were searched for papers containing the key words "lateral meniscus posterior root tears", "LMPRTs" and "risk factor". Inclusion screening, data extraction, and quality assessment of the included articles were conducted independently by two authors. Statistical analysis was conducted to determine risk factors for LMPRTs. RESULT: Seventeen studies with a total sample size of 6, 589 patients were identified. The pooled prevalence of LMPRTs was 9.6% (range, 5.1-33.8%) for ACL injury. Significant risk factors included a patient age of < 30 [OR = 1.4, 95% CI (1.07, 1.84), p = 0.01], male sex [OR = 1.50, 95% CI (1.24,1.81), p = 0.01], higher body mass index (BMI) [MD = 0.45, 95% CI (0.13, 0.76), p < 0.01], higher lateral posterior tibial slope (LPTS) [MD = 2.22, 95% CI (1.37, 3.07), p < 0.01], deep sulcus sign [OR = 5.76, 95% CI (1.35, 24.52), p < 0.01] and bone bruises on lateral femoral condyle [OR = 4.88, 95% CI (1.27, 18.77), p < 0.01], lateral meniscal extrusion > 1 mm [OR = 5.56, 95% CI (1.52, 20.29), p < 0.01] and > 3 mm [OR = 12.91 95% CI (1.28, 130.01), p < 0.01], medial meniscal tears [OR = 1.40, 95% CI (1.12, 1.75), p < 0.01], and medial ramp lesions [OR = 2.29, 95% CI (1.35, 3.89), p < 0.01]. CONCLUSION: Age below 30, male, higher BMI, higher LPTS, deep sulcus sign, bone bruises on lateral femoral condyle, lateral meniscal extrusion, medial meniscal tear, and medial ramp lesion are risk factors for LMPRTs. LEVEL OF EVIDENCE: Level IV.

The Protective Effect of Tetrahydroxystilbene Glucoside on High Glucose-Induced Injury in Human Umbilical Vein Endothelial Cells through the PI3K/Akt/eNOS Pathway and Regulation of Bcl-2/Bax.

Endothelial dysfunction plays a central role in the patho-genesis of diabetic vascular complications. 2,3,5,4'-tetra-hydroxystilbene-2-O-ß-D-glucoside (TSG), an active component extracted from the roots of Polygonum multiflorum Thunb, has been shown to have strong antioxidant and antiapoptotic activities. In the present study, we investigated the protective effect of TSG on apoptosis induced by high glucose in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms. Our data demonstrated that TSG significantly reversed the high glucose-induced decrease in cell viability, suppressed high glucose-induced generation of intracellular reactive oxygen species (ROS), the activity of caspase-3, and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, we found that TSG not only increased the expression of Bcl-2, while decreasing Bax expression, but also activated phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) with subsequent nitric oxide production and ultimately reduced high glucose-induced apoptosis. However, the antiapoptotic effects of TSG were abrogated by pretreatment of the cells with PI3K inhibitor (LY294002) or eNOS inhibitor NG-L-nitro-arginine methyl ester, respectively. These results suggest that TSG inhibits high glucose-induced apoptosis in HUVECs through inhibition of ROS production, activation of the PI3K/Akt/eNOS pathway, and upregulation of the Bcl-2/Bax ratio, and thus may demonstrate significant potential for preventing diabetic cardiovascular complications.

Identification and characterization of melon circular RNAs involved in powdery mildew responses through comparative transcriptome analysis.

Circular RNAs (circRNAs) are a class of newly discovered non-coding RNAs that are typically derived from a genome's exonic, intronic, and intergenic regions. Recent studies of circRNAs in animals and plants have shown that circRNAs are vital in response to various abiotic and biotic stresses. Powdery mildew disease (PM) is a serious fungal disease threatening the melon industry. We performed whole transcriptome sequencing using the leaves of a PM-resistant (M1) and a PM-susceptible (B29) melon to identify circRNAs and determine their molecular functions. A total of 303 circRNAs were identified and >50% circRNAs were derived from exonic regions. Expression levels were significantly altered in 17 and 23 circRNAs after PM infections in B29 and M1, respectively. Melon circRNAs may participate in the response to biotic stimuli, oxidation reduction, metabolic processes, and the regulation of gene expression based on the functional annotation of circRNA parental genes. Furthermore, 27 circRNAs were predicted to be potential targets or 'sponges' for 18 microRNAs (miRNAs). Our results are the first to identify and characterize circRNA functions in melon and may contribute to a better understanding of the role and regulatory mechanisms of circRNAs in resisting PM.

Vitamin D Decreases Plasma Trimethylamine-N-oxide Level in Mice by Regulating Gut Microbiota.

As a metabolite generated by gut microbiota, trimethylamine-N-oxide (TMAO) has been proven to promote atherosclerosis and is a novel potential risk factor for cardiovascular disease (CVD). The objective of this study was to examine whether regulating gut microbiota by vitamin D supplementation could reduce the plasma TMAO level in mice. For 16 weeks, C57BL/6J mice were fed a chow (C) or high-choline diet (HC) without or with supplementation of vitamin D3 (CD3 and HCD3) or a high-choline diet with vitamin D3 supplementation and antibiotics (HCD3A). The results indicate that the HC group exhibited higher plasma trimethylamine (TMA) and TMAO levels, lower richness of gut microbiota, and significantly increased Firmicutes and decreased Bacteroidetes as compared with group C. Vitamin D supplementation significantly reduced plasma TMA and TMAO levels in mice fed a high-choline diet. Furthermore, gut microbiota composition was regulated, and the Firmicutes/Bacteroidetes ratio was reduced by vitamin D. Spearman correlation analysis indicated that Bacteroides and Akkermansia were negatively correlated with plasma TMAO in the HC and HCD3 groups. Our study provides a novel avenue for the prevention and treatment of CVD with vitamin D.

Comparative transcriptome analysis uncovers regulatory roles of long non-coding RNAs involved in resistance to powdery mildew in melon.

BACKGROUND: Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with more than 200 nucleotides in length, which play vital roles in a wide range of biological processes. Powdery mildew disease (PM) has become a major threat to the production of melon. To investigate the potential roles of lncRNAs in resisting to PM in melon, it is necessary to identify lncRNAs and uncover their molecular functions. In this study, we compared the lncRNAs between a resistant and a susceptible melon in response to PM infection. RESULTS: It is reported that 11,612 lncRNAs were discovered, which were distributed across all 12 melon chromosomes, and > 85% were from intergenic regions. The melon lncRNAs have shorter transcript lengths and fewer exon numbers than protein-coding genes. In addition, a total of 407 and 611 lncRNAs were found to be differentially expressed after PM infection in PM-susceptible and PM-resistant melons, respectively. Furthermore, 1232 putative targets of differently expressed lncRNAs (DELs) were discovered and gene ontology enrichment (GO) analysis showed that these target genes were mainly enriched in stress-related terms. Consequently, co-expression patterns between LNC_018800 and CmWRKY21, LNC_018062 and MELO3C015771 (glutathione reductase coding gene), LNC_014937 and CmMLO5 were confirmed by qRT-PCR. Moreover, we also identified 24 lncRNAs that act as microRNA (miRNA) precursors, 43 lncRNAs as potential targets of 22 miRNA families and 13 lncRNAs as endogenous target mimics (eTMs) for 11 miRNAs. CONCLUSION: This study shows the first characterization of lncRNAs involved in PM resistance in melon and provides a starting point for further investigation into the functions and regulatory mechanisms of lncRNAs in the resistance to PM.

The mirid bug Apolygus lucorum deploys a glutathione peroxidase as a candidate effector to enhance plant susceptibility.

The mirid bug Apolygus lucorum has become a major agricultural pest since the large-scale cultivation of Bt-cotton. It was assumed that A. lucorum, similarly to other phloem sap insects, could secrete saliva that contains effector proteins into plant interfaces to perturb host cellular processes during feeding. However, the secreted effectors of A. lucorum are still uncharacterized and unstudied. In this study, 1878 putative secreted proteins were identified from the transcriptome of A. lucorum, which either had homology with published aphid effectors or shared common features with plant pathogens and insect effectors. One hundred and seventy-two candidate effectors were used for cell death-inducing/suppressing assays, and a putative salivary gland effector, Apolygus lucorum cell death inhibitor 6 (Al6), was characterized. The mRNAs of Al6 were enriched at feeding stages (nymph and adult) and, in particular, in salivary glands. Moreover, we revealed that the secreted Al6 encoded an active glutathione peroxidase that reduced reactive oxygen species (ROS) accumulation induced by INF1 or Flg22. Expression of the Al6 gene in planta altered insect feeding behavior and promoted plant pathogen infections. Inhibition of cell death and enhanced plant susceptibility to insect and pathogens are dependent on glutathione peroxidase activity of Al6. Thus, this study shows that a candidate salivary gland effector, Al6, functions as a glutathione peroxidase and suppresses ROS induced by pathogen-associated molecular pattern to inhibit pattern-triggered immunity (PTI)-induced cell death. The identification and molecular mechanism analysis of the Al6 candidate effector in A. lucorum will provide new insight into the molecular mechanisms of insect-plant interactions.

The glycoside hydrolase 18 family chitinases are associated with development and virulence in the mosquito pathogen Pythium guiyangense.

Chitinases, the enzymes responsible for the biological degradation of chitin, participate in numerous physiological processes such as nutrition, parasitism, morphogenesis and immunity in various organisms. However, the genome-wide distribution, evolution and biological functions of chitinases are rarely reported in oomycetes. This study systematically investigated the glycoside hydrolase 18 (GH18) family of chitinases from the mosquito pathogenic oomycete, Pythium guiyangense using bioinformatics and experimental assays. A total of 3 pairs of GH18 chitinase genes distributed in three distinct phylogenic clusters were identified from P. guiyangense genome, which is consistent with the ones in plant pathogenic oomycetes. Further transcriptional analysis revealed that Pgchi1/2 was highly expressed at the development stages, while Pgchi3/4 and Pgchi5/6 were up-regulated at the infection stages. The biological function analysis of chitinase genes using genetic transformation silencing method showed that silencing of Pgchi1/2 resulted in reduced zoospore production, without affecting the virulence. However, attenuation of Pgchi3/4 and Pgchi5/6 genes regulated not only oxidative stress responses, but also led to decreased infection rates to mosquito larvae. Taken together, this study provides a comprehensive overview of P. guiyangense chitinase family and reveals their diverse roles in the development, stress response, and virulence, which would elucidate insightful information on the molecular mechanism of chitinase in entomopathogenic pathogens.

Genome-wide and functional analyses of tyrosine kinase-like family genes reveal potential roles in development and virulence in mosquito pathogen Pythium guiyangense.

The tyrosine kinase-like (TKL) gene family is widely existed in most eukaryotes and participates in many biological processes, however, has been rarely studied in oomycetes. In this study we performed bioinformatic and experimental analyses to characterize TKLs in Pythium guiyangense, a promising mosquito biological control agent. Our results revealed that TKLs were widely distributed in all the detected oomycetes, but were largely expanded in P. guiyangense in a species-specific expansion manner. The expansion was mostly driven by whole-genome duplication and tandem duplication. Domain distributions and exon-intron structures were highly conserved in the same group while diverse in different groups, suggesting of functional divergence. Transcriptional analysis revealed that over one fourth of TKLs were differentially expressed after infection of mosquito larvae, implying that these genes might participate in the infection process. Furthermore, subgroup A TKLs were functionally investigated using genetic transformation silencing method. Our findings demonstrated that subgroup A TKLs were up-regulated at the early infection stages and silencing of subgroup A TKLs led to reduced mycelia growth, zoospore production and alteration of stress responses. Pathogenicity assays also revealed that silencing of subgroup A TKLs reduced P. guiyangense virulence to mosquito larvae. Taken together, this study provides a comprehensive overview of P. guiyangense TKL family and reveals their potential roles in growth, development, stress response, and especially virulence.