| Bursaphelenchus mucronatus is a parasitic nematode of pine that is widely distributed in the natural pine forests of Asia and Europe.Its morphology and biology are similar with Bursaphelenchus xylophilus,the causal agent of pine wilt disease.Both species feed on epithelial cells and reproduce in the resin canals of living pine trees,and occupy the same niche space.B.mucronatus has never been demonstrated to be associated with epidemics of pine wilt disease in its known areas of distribution over the past 30 years.However,over the past decade,several studies have demonstrated that the virulence of different B.mucronatus isolates varies to some extent.To date,relative researches of B.mucronatus focuses on their differences with B.xylophilus,rare on the protein and gene level of themselves.In the present study,the types,functions and number of differentially expressed genes of different virulence B.mucronatus isoltaes during the pathogenic processes were analysed through the technology of transcriptome and proteome,and several important differential proteins invovled in the pathogenic processes were identified,which increases our understanding of B.mucronatus pathogenesis.The main results and conclusions are as follows.1.To investigate the virulence of B.mucronatus,2-year-old seedlings of Pinus massoniana and Pinus elliottii were inoculated with one of six isolates of B.mucronatus under field conditions.The virulence of the six B.mucronatus isolates differed on the three host species: P.massoniana seedling mortality ranged from zero to six of the 18 inoculated seedlings whereas P.elliottii seedling mortality ranged from 22.2% to 83.3%.Three B.mucronatus isolates that appeared to cause different levels of mortality among the seedlings were used to inoculate12-year-old Pinus thunbergii trees.The trees were monitored for a year,during which time between 22.2% and 66.7% of the inoculated trees in each treatment wilted and died.The average monthly temperature during the test period appeared to be similar to that of the historical average in the test areas;however,both study sites experienced above average rainfall.This study demonstrated that B.mucronatus has potential virulence on pine trees and provided experimental evidence that high temperatures or drought stress are not essential for the virulence of B.mucronatus.2.Sixty-one isolates of B.mucronatus collected from 14 Chinese provinces,Japan and Korea were used for ISSR analysis.An unweighted pair group cluster analysis dendrogram clustered them as two independent clades.Principal component analysis showed the phylogenetic relationship of the nematode isolates more clearly;isolates were separated into more than four groups.The results of the Mantel test indicated correlation of genetic distance matrices and geographic distance matrices were significant(p < 0.05,r = 0.312).The genetic differentiation coefficient and gene flow of B.mucronatus were 0.341 and 1.091,respectively,suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B.mucronatus isolates in China.3.Real-time fluorescent quantitative PCR analysis is a powerful analytical technique,but depend on the use of stable reference genes.This study proposed to test the expression stability of 8 candidate reference genes(alpha-actin,elongation factor 1-alpha,histone,peroxisomal membrane protein,beta-tubulin,ubiquitin-conjugating enzyme,ubiquitin and 18 S ribosomal RNA)in B.mucronatus by five algorithms,geNorm,NormFinder,BestKeeper,deltaCq and RefFinder.Gene expression stability of these genes was assayed across different habitat and developmental stages of B.mucronatus.In general,our results showed beta-tubulin,18 S ribosomal RNA and ubiquitin-conjugating enzyme as the most stable genes in different habitat conditions,while beta-tubulin,histone and 18 S ribosomal RNA as the most stable genes in different developmental stages.Hence,beta-tubulin and18 S ribosomal RNA were the most stable reference genes in different habitat and developmental stages of B.mucronatus.This study provides a list of reliable reference genes for transcript normalization in B.mucronatus in different habitat and developmental stages,which will facilitate genetic studies in this nematode.4.We conducted an integrated study coupling RNA sequencing and isobaric tags for relative and absolute quantitation to analyse transcriptomic and proteomic data of highly and weakly virulent B.mucronatus isolates during the pathogenic processes.Approximately 40,000 annotated unigenes and 5000 proteins were gained from the isolates.When we matched all of the proteins with their detected transcripts,a low correlation coefficient of r = 0.138 was found,indicating probable post-transcriptional gene regulation involved in the pathogenic processes.A functional analysis showed that five differentially expressed proteins which were all highly expressed in the highly virulent isolate were involved in the pathogenic processes of nematodes.Peroxiredoxin,fatty acid-and retinol-binding protein,and glutathione peroxidase relate to resistance against plant defence responses,while ?-1,4-endoglucanase and expansin are associated with the breakdown of plant cell walls.Thus,the pathogenesis of B.mucronatus depends on its successful survival in host plants.5.Base on the RNA-Seq of B.mucronatus,the full-length cDNA of fatty acid-and retinol-binding protein and calreticulin genes from B.mucronatus were cloned by rapid-amplification of cDNA ends,694 and 1630 bp,respectively.The complete fatty acid-and retinol-binding protein gene open reading frame is 537 bp,and encodes a polypeptide with 178 amino acid(the protein molecular weight is 19601.3),while the complete calreticulin gene open reading frame is 1209 bp,coding a polypeptide with 402 amino acid(the protein molecular weight is 46866.72).To confirm their involvement in B.mucronatus' virulence,fatty acid-and retinol-binding protein gene and calreticulin gene were used for construction RNA interference.Once the RNA interference were constructed and introduced into the nematode,RT-qPCR was used to assess the fatty acid-and retinol-binding protein gene and calreticulin gene mRNA expression levels in different treatments.By comparing the expression levels of the fatty acid-and retinol-bindingprotein gene in the nematodes exposed to the target dsRNA-soaking solution and controls,it was shown that the RNA interference procedure caused a significant reduction in the target mRNA's expression level.Taking the mRNA expression level in M9 medium(non-dsRNA treated)as100%,the average expression levels of samples with the green fluorescent protein gene dsRNA treatment was 99.8%,while with the target dsRNA treatment it was only 26.9%.The expression levels of the calreticulin gene produced similar results after soaking in an calreticulin gene dsRNA solution.The number of dsRNA-treated nematode offspring was used to assess the influence of RNAi on the development and propagation of B.mucronatus.The number of offspring from the dsRNA-treated nematodes was less than that of non-dsRNA-treated nematodes.In the non-dsRNA treatment,an average of 1239 individuals in the F1 generation were produced by 20 pairs of females and males,but an average of only 945 and 906 offspring were produced in the dsRNA treatments of fatty acid-and retinol-binding protein and calreticulin gene,respectively.The difference between the target dsRNA treatment and M9 was highly significant(p-value<0.001).Treatment with green fluorescent protein gene dsRNA soaking resulted in an average of 1133 individuals in the F1 generation,which is a little less than that of non-dsRNA treatment,but not highly significant.However,it is highly significantly different(p-value<0.001)from the target dsRNA treatment.Thus,the knockdowns of the two target genes had marked effects on the development and propagation of B.mucronatus.Additionally,a possible dsRNA toxicity to the nematodes was not obvious at a concentration of 800 ng/?L.To assess the influence of RNA interference on the virulence of B.mucronatus,the dsRNA-treated nematodes were inoculated on two-year-old Pinus thunbergii seedlings under greenhouse conditions.The first wilted seedling infected with fatty acid-and retinol-binding protein gene dsRNA-,green fluorescent protein gene dsRNA-,and M9-treated nematodes were observed 5weeks after inoculation,and the first wilted calreticulin gene dsRNA-treated seedling was observed 6 weeks after inoculation.All of the treatments of B.mucronatus caused some seedlings to wilt;while the atty acid-and retinol-binding protein and calreticulin gene dsRNA-treated nematodes did not cause as many wilted seedlings as the GFP dsRNA-and M9-treated. |
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