Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis

Abstract

Pythium guiyangense , an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P . guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P . guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P . guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P . guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P . guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P . guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P . guiyangense effectors might also be useful for management of other important agricultural pests.

Prediction and Characterization of RXLR Effectors inPythiumSpecies

“…Our previous synteny analysis showed that P. guiyangense has a hybrid genome derived from two distinct parental species, which leads to a nearly two-fold expansion of its predicted genes when compared with other Pythium species. Consequently, most homology gene pairs in P. guiyangense show high degrees of collinearity [24]. However, we only found 4 RXLRs from different scaffolds showing collinearity, indicating that additional mechanisms may also contribute to the expansion of RXLR genes in P. guiyangense .…”

“…No Pythium RXLR effector has been previously identified despite the numerous reports of RXLRs in their phylogenetically close species [21,22,23,24]. Here we developed a de novo identification method with stringent threshold to screen RXLR candidates in 9 Pythium species (Fig 1A).…”

“…The genome sequences of H. arabidopsidis [28], Ph. vexans [22], P. aphanidermatum [22], P. arrhenomanes [22], P. irregulare [22], P. iwayamai [22], P. oligandrum [39], P. periplocum [39], P. ultimum [21], P insidiosum [23], P. guiyangense [24], S. parasitica [40], P. tricornutum [41], T. pseudonana [42], S. cerevisiae [43], S. sclerotiorum [44], U. maydis [45], P. graminis [46], B. cinerea [47], C. elegans [48], M. hapla [49], E. coli [50], E. amylovora [51] and Ca L. asiaticus [52] were obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). Detailed genomic information of all species is listed in S7 Table.…”

“…All these RXL-containing oomycetes are phylogenetically close to Pythium species. Nevertheless, previously reported Pythium genome analyses did not reveal any RXLR-encoding genes [21,22,23,24].…”

“…Interestingly, Some Pythium species, such as P. oligandrum and P. periplocum , are used as mycoparasitic biocontrol agents since they can infect fungal hosts and induce plant defense response as well [25,26,27]. Likewise, P. guiyangense is a parasite used for mosquito control [24].…”