| Sitobion avenae,as the dominant species of wheat aphids in various wheat regions of China,has differentiated into different biotypes that can overcome the resistance of cereal crop varieties.This has seriously affected the development and application of aphid-resistant cereal crop varieties.Therefore,it is of great significance to elucidate the differentiation mechanism of this aphid biotypes.Buchnera aphidicola is a primary symbiotic bacterium in most aphids,which participates in the host’s amino acid metabolism and can play an important role in the biotype differentiation of aphids.However,the effects and mechanism of this bacterium on the biotype differentiation of S.avenae are still unclear.Based on RNA-Seq,our team’s previous research provided evidence for the involvement of Buchnera in the biotype differentiation of S.avenae,and also provided important candidate genes and target amino acid metabolic pathways for this study to further explore the mechanism of Buchnera-mediated biotype differentiation.On this basis,the project aims to explore the role of Buchnera in the biotype differentiation of S.avenae,and to clarify the amino acid metabolic pathways and key genes involved in the in the biotype differentiation of this aphid mediated by Buchnera.The main findings are as follows:1.Using qRT-PCR,we detected thedifferences in Buchnera abundance in six biotypes of Sitobion avenae at the 5-day old nymphs and adult stages,while feeding on six host varieties.The results showed that there were significant differences in Buchnera abundance of the same biotype when feeding on different host varieties.We also found significant differences in Buchnera abundance existed among different biotypes feeding on the same variety.The abundance of Buchnera in 5-day old nymphs was significantly higher than that in adults.Furthermore,for biotype 1,biotype 2,and biotype 4 of S.avenae,there was a significant positive correlation between the fecundity and the Buchnera abundance.The "host plant","biotype","aphid stage(5-d old nymphs and adult)" and their interactions all significantly affected the abundance of Buchnera.2.The antibiotic rifampicin was added to full nutrition artificial diets to reduce the Buchnera abundance,and the differences in 10-day fecundity and the damage performance of six biotypes on different barlry/wheat varieties(i.e.,Mingxian 169,Zhong 4 Wumang,186-TM12-34,Dulihuang,Zaoshu No.3,Xiyin No.2)were detected.The results indicated that rifampicin had a significant effcet on the Buchnera abundance and fecundity of S.avenue.The Buchnera abundance and fecundity were negatively correlated with rifampicin concentration,and the range of change gradually slowed down with increasing concentration of rifampicin.When the concentration of rifampicin was 2 μg/mL,the Buchnera abundance decreased significantly and the aphids had a certain fecundity,which could be used for subsequent detection of the damage performance on different host plants for S.avenae biotypes.Before rifampicin treatment,there were differences in host damage performance among the six biotypes,and significant differences in fecundity among the six barley/wheat varieties were detected.After rifampicin treatment,the 10-d fecundity of six biotypes on six barley/wheat varieties decreased significantly.In addition,the fecundity on the susceptible variety Mingxian 169 was significantly higher than that of the five resistant varieties(i.e.,Zhong 4 Wumang,186-TM12-34,Dulihuang,Zaoshu No.3,Xiyin No.2),and the host damage performance of six biotypes tended to be similar on the five resistant varieties.3.Based on the results of RNA-Seq analysis,with full nutrition artificial diet as control,the target amino acids(i.e.,leucine,tryptophan,valine,and isoleucine)were removed respectively,and the effects of the lack of these essential amino acids on the developmental duration and fecundity of six S.avenae biotypes were studied,in order to clarify the key amino acid synthesis pathways of Buchnera-mediated biotype differentiation.The results showed that the removal of Leu,Val,Trp,or Ile had significant effects on the developmental duration of nymphs and the 10-day fecundity of each S.avenae biotype.Furthermore,the responses of each biotype to the lack of amino acids differed.Principal component analysis results showed Leu and Trp had the highest correlation with the total developmental duration of nymphs and 10-d fecundity of S.avenae,respectively.This suggests the metabolic pathways of these two amino acids may play a crucial role in the biotype differentiation mediated by Buchnera,Therefore,the expression differences of related amino acid synthesis genes(i.e.,Isopropylmalate dehydratase-LeuB,Anthranilate synthase TrpEG,obtained by RNA-Seq)were determined in the six biotypes when feeding with Leu-free,Trp-free,and full nutrition artificial diet.On the pure artificial diet,there were significant differences in the relative expression levels of LeuB and TrpEG in each biotype of Buchneria.On the full nutrition artificial diet,the relative expression levels of LeuB and TrpEG in each biotype were significantly different.After feeding on Leu-free,Trp-free diets,the LeuB and TrpEG genes were both up-regulated compared with the full nutrition dites control,and the up-regulation range was different for different biotypes(1.67-4.12 times)..In summary,Buchnera plays an important role in mediating the biotype differentiation of S.avenae.The difference in Buchnera abundance when feeding on different wheat varieties may be one of the reasons for the differences in the fitness(host damage performance)of the same biotype on different host varieties.Leu and Trp may be the key nutrient metabolic pathways for the biotype differentiation of S.avenae mediated by Buchnera.For different biotypes,there were differences in the ability of Buchnera to synthesize these two amino acids.These results preliminarily reveal the crucial role and mechanism of Buchnera in mediating biotype differentiation and regulating host damage performance for S.avenae.This provides a research basis for further exploration of molecular mechanisms,and can provide new theoretical basis for exploring host-symbiotic interactions and breeding of aphid-resistant cereal crops. |
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