Project description:RNA was extracted from a pooled sample of salivary glands that were excised from adult Diuraphis noxia aphids of two biotypes SA1 and SAM2.0. South African D. noxia biotype SA1 is known to be the least virulent aphid, while its offspring, the South African D. noxia biotype SAM2.0 is a newly developed biotype that is capable of feeding on several resistant cultivars. The overall purpose of the experiment was to establish a baseline availability of transcripts contained in the salivary glands of D. noxia aphids as well as help improve on current genome assemblies. In total, 200 glands were excised from both biotypes feeding on various wheat hosts (Tugela, Tugela Dn1, Gamtoss R, SST387). Aphid glands were excised in PBS buffer and stored in RNA-later until RNA extraction with a Qiagen Rneasy kit could commence. Library preparation for sequencing was performed using an Illumina TruSeq Stranded mRNA LT Sample Prep Kit following the TruSeq Stranded mRNA Sample Preparation Guide, Part # 15031047 Rev. E protocol. The single library was then sequenced on a NovaSeq6000 system. De novoassembly was performed making use of the Trinity package.
Project description:BackgroundProteins within aphid saliva play a crucial role as the molecular interface between aphids and their host plants. These salivary effectors modulate plant responses to favour aphid feeding and facilitate infestation. The identification of effectors from economically important pest species is central in understanding the molecular events during the aphid-plant interaction. The Russian wheat aphid (Diuraphis noxia, Kurdjumov) is one such pest that causes devastating losses to wheat and barley yields worldwide. Despite the severe threat to food security posed by D. noxia, the non-model nature of this pest and its host has hindered progress towards understanding this interaction. In this study, in the absence of a salivary gland transcriptome, whole-body transcriptomics data was mined to generate a candidate effector catalogue for D. noxia.ResultsMining the transcriptome identified 725 transcripts encoding putatively secreted proteins amongst which were transcripts specific to D. noxia. Six of the seven examined D. noxia putative effectors, termed DnE's (Diuraphis noxia effectors) exhibited salivary gland-specific expression. A comparative analysis between whole-body D. noxia transcriptome data versus the head and body transcriptomes from three other aphid species allowed us to define a catalogue of transcripts putatively upregulated in D. noxia head tissue. Five of these were selected for RT-qPCR confirmation, and were found to corroborate the differential expression predictions, with a further three confirmed to be highly expressed in D. noxia salivary gland tissue.ConclusionsDetermining a putative effector catalogue for D. noxia from whole-transcriptome data, particularly the identification of salivary-specific sequences potentially unique to D. noxia, provide the basis for future functional characterisation studies to gain further insight into this aphid-plant interaction. Furthermore, due to a lack of publicly available aphid salivary gland transcriptome data, the capacity to use comparative transcriptomics to compile a list of putative effector candidates from whole-body transcriptomics data will further the study of effectors in various aphid species.
Project description:Phenotypic responses to biotic stresses are often studied as the interactions between two species; however, in the phytobiome, these responses frequently result from complex interactions involving several organisms. Here, we show that variation in chlorosis caused by Russian wheat aphid (Diuraphis noxia) feeding is determined, in part, by aphid-associated bacteria. Proteomic analysis of fluids injected into a sterile medium by the aphid during feeding indicate that 99% of the proteins are of bacterial origin. Of these, the greatest proportion are produced by bacteria in the order Enterobacteriales. Bacteria from five genera in four families that have the capacity to produce these proteins were isolated directly from aphids as well as from wheat leaves only after D. noxia feeding. By themselves or in combination, these bacteria were not virulent to wheat, even at high inoculum levels. Metagenomic analysis showed that the same five D. noxia-associated genera dominated the non-Buchnera component of the aphid microbiome, and that representation of these genera was reduced in aphids from colonies established after isolation of newborn nymphs from their mothers prior to feeding (‘isolated’ aphids). Isolation or treatment with antibiotics reduced bacterial numbers, and these aphids caused less feeding damage on wheat than non-isolated or non-antibiotic treated aphids. Our data show that bacterial proteins are a significant component of Russian wheat aphid saliva, that the bacteria producing these proteins are associated with aphids and plants fed upon by aphids, and that these aphid-associated bacteria facilitate aphid virulence to wheat.