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:RNA was extracted from all instar (insect developmental) stages for both D. noxia biotypes SA1 and SAM with the purpose to capture as many expressed transcripts as possible. South African D. noxia biotype SA1 is known to be the least virulent aphid, while its offspring, the South African D. noxia biotype SAM is the most virulent. The overall purpose of the experiment was to establish a baseline availability of transcripts to the aphids as well as help improve on current genome assemblies. Three biological replicates of 100 aphids each was collected from both biotypes SA1 and SAM that were respectively reared on preference host cultivars. Whole aphids were flash frozen in liquid nitrogen, ground to a powder with micro pistils and RNA was extracted making use of a Qiagen RNeasy kit. 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 replicate samples from the SAM biotype yielded between 120 – 140 million 100bp PE reads and the replicate samples from the SA1 biotype yielded between 113 – 137 million 100bp PE reads (with a Q20 phred score above 98% for all replicates) after sequencing on the NovaSeq6000 system. De novoassembly was performed making use of the Trinity package.
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.
