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.

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.

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.

New discoveries and applications of mosquito fungal pathogens.

Mosquitoes are a major threat to human health globally because they transmit infectious diseases, such as malaria, lymphatic filariasis, and arboviruses. The conventional mosquito control efforts, based on synthetic insecticides, have been compromised owing to the eventual development of insecticide resistance and the adverse environmental impacts of insecticides. Alternative eco-friendly approaches using entomopathogenic fungi to alleviate vector-borne disease burden have gained an increasing interest because of their selective specificity and environmental safety. Existing literature revealed an enormous potential of microbial agents for the biocontrol of mosquitoes. With the advances in genetic recombination and transformation techniques, genetically engineered fungal biopesticides showed promising efficacy against insecticide-resistant mosquitoes. In this article, we elaborate on the important mosquito fungal and oomycota pathogens as potential biocontrol agents and infection mechanism through oral ingestion. Recent advances on the secreted effectors for suppression of host immunity and progress on the development of transgenic mosquito-killing fungi were discussed.