| Pine wilt disease（PWD） is a destructive disease upon pine trees. Due to its rapid spreading rate, the disease has drawn more and more attention all over the world. Pine wood nematode（PWN）, Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. Although there are several hypotheses about the mechanism of PWD, such as cellulase, phytotoxin and terpenoid hypotheses, the pathogenic mechanism of PWD still remains to be elucidated. Micro RNAs（mi RNAs） are a set of short（21nt）, endogenous RNAs that play crucial regulatory roles in both animals and plants. Currently, the study on mi RNAs mainly involved in humans and other model organism and bioinformatics analyses suggest that over one-third of human genes may be targets of mi RNAs, most of which are transcriptional and developmental factors. Moreover, abnormal mi RNA expression would potentially leads to human disease and even death in the case of severe phenotypes. However, the relative mi RNAs study in B. xylophilus was not well conducted. But the investigation on mi RNAs may be a critical step in facilitating our understanding of interaction between PWNs and pine trees. Thus, we use deep sequencing technique to describe the dynamic changes in mi RNAs expression during the pathological process of pine wilt disease.It is reported that two forms of PWNs existed in its native region, i.e., strongly virulent（SV） and weakly virulent（WV）. Some previous studies proved that the lower reproductivity and increased developmental time of a generation were observed in weakly virulent strains rather than strongly virulent strains. Other studies indicated PWNs with different virulence contained different enzymatic and non-enzymatic molecules which were involved in oxidative stress metabolism. Usually, the virulence of PWN was evaluated by an inoculation test and some research also reported other classification method based on PCR-RFLP patterns of heat-shock protein 70 A. However, genome wide identifications of the molecular variances between strongly and weakly virulent nematode strains have not been reported yet. With the help of high-throughput sequencing technique, the transcriptome level comparisons between PWNs with diffferent virulence（SV: AA3,AMA3 and ZL1; WV: YW4） were performed to identified genes and exons with significant changes. Functional annotations indicated some of the differentially expressed genes could potentially contributed to the virulence variations existed between the two types of nematodes. Also, genome wide identification of SNPs revealed the global SNPs variations between the two PWNs forms and identified 117 SNPs which could be used to evaluate the virulence of PWNs other than traditional inoculation tests. All the main results were conveyed as follows:（1）We obtained 17, 94, 104, and 85 evolutionarily conserved mi RNAs from Control（PWNs grow on fungi）, F（first stage of PWD）, M（middle stage of PWD） and L（last stage of PWD） libraries, respectively. Four mi RNAs, mi R-1, mi R-71, mi R-100 and let-7, were detected to be expressed in dominant abundance in all libraries. Compared with previous mi RNAs study on PWN, our research identified 97 more evolutionary conserved mi RNA.（2） Here, we found 129, 221, 50, and 74 novel mi RNAs from Control, F, M and L stages, respectively. Generally, 256 novel unique mi RNA families were detected and assigned with a temporal gene ID. Of these 256 novel families, only 19 were expressed throughout all stages. Ten novel mi RNAs were found to be expressed solely in the pathological process of PWD. Relatively few mi RNAs, like bxynovelcandidate1, bxynovelcandidate2, and bxynovelcandidate3, were in fact found to be highly expressed in comparison with previously identified mi RNAs.（3）We grouped the conserved mi RNAs expression into four sub clusters.. For instance, only several mi RNAs like mi R-100, mi R-50, mi R-87, mi R-86 was found highly expressed only in Control stage. Mir-1 and mi R-57 reached their peak in F stage. Mi R-58, mi R-2226, mi R-48 and mi R-45 exhibited a huge boost and reached their peaks in the M stage, followed by a steep decline in L stage. Besides, a few mi RNAs like mi R-2, mi R-34,mi R-71 showed a increase in the L stage. All of these facts were further confirmed by the subsequent recovery of PWNs from the three infection stages in this research. The population of PWNs were most abundant in the M stage but decreased in the L stage. These phenomenons were inaccordance with the mi RNAs expression patterns and it is possible that mi RNAs played important roles during the pathogenic process of PWD.（4） A compelling discovery was that many of targets were enriched in hydrolase, transferas activity and nematode development. Based on the fact that strict complementarity between mi RNAs and their targets,we found only two mi RNAs, mi R-73 and mi R-239, as desired mi RNAs since they exhibited perfect 5′end base pairing with GH45 cellulase homologs. Therefore, mi RNAs could play important rols in PWD by facilitating the invasion of PWNs as well as breaking down the plant cell walls.（5） A non-redundant transcript assembly was generated by merging all four assemblies together. Eventually, we got 20,465 transcripts derived from 17,199 genes. The length distributions of assembled transcripts mostly ranged from 100 bp to 2.5kb, and seven transcripts were identified with their length over 20 kb. Previous research on the B. xylophilus transcriptome reported a transcript assembly comprised 23,765 m RNA contigs with maximum length of 5,847 using de-novo methods. Compared with the early de-novo assembly, our genome-based transcript assembly reduced the transcripts number to 20,465 and increased the maximal length to 24,372. In addition, there were 904 transcripts with lengths longer than 5847.（6） Finally, 238 overlapped DE transcripts were identified using the two packages together（Pvalue<0.05）. The hierarchy clustering results of DE transcripts showed that SV strains were grouped together rather than the WV strain. Functional annotation indicated that differentially expressed transcripts were mainly involved with growth regulation and oxidoreductase activity. This discovery supported the fact that different nematode virulence strains may have different growth abilities. Recent studies indicated that more than ten anti-oxidant proteins were identified in the secretome of PWNs. The secreted anti-oxidant enzymes would play an important role in protecting PWNs itself from oxygen free radicals in the pine tree.（7） Expression profiling of exons revealed that there were 75 genes containing 84 DE exons. Meanwhile, 63, 65, 53 and 56 exon-skipping candidates were identified from AA3, AMA3, ZL1 and YW4, respectively, using RNA-seq alone. Among them, 35 recurrent exon-skipping candidates were detected in all nematode strains. GO enrichment analysis indicated transcripts contained in those DE genes were mainly associated with functions like: growth rate regulation and larval development.（8）A total of 362563 were identified from the four nematode strains. To be more specific, there were 362232（homozygote:352323;heterozygote:9909） SNPs found in AA3, 362226（homozygote:352462;heterozygote:9764） SNPs found in AMA3, 362157 SNPs（homozygote:352522;heterozygote:9635） found in ZL1 and 362221（homozygote:352219;heterozygote:10002） SNPs found in YW4. Among all those SNPs, the most dominant genotypes were G->A, A->G, C->T and T->C. In addition, more than 95% of the SNPs were homozygous and more than 60% of the SNPs were located within intergenic and intron regions.（9） 117 SNPs were selected as possible markers to distinguish these two forms based on genotype variations. These 117 SNPs shared the same genotype among SV but exhibited different genotypes in WV. In addition, 45 of them were located in exons and 72 of them located in introns. Particularly, we found there were four SNPs which underwent post-transcriptional RNA editing in SV strains only. These editing events ensured that mi R-47 could successfully recognize their target genes. It indicated that RNA editing could couple with mi RNA to execute different post-transcriptional regulation of B. xylophilus with different virulence.This study provided comprehensive descriptions upon the dynamic change of mi RNAs during the pathological process of PWD, revealed the molecular variations between PWNs with different virulence and established a sub set of SNPs markers for rapid detection of nematodes virulences. All the results inevitably enhanced our understanding towards the mechanism of PWD and provided fundamental resources for preventing and controlling this devastating disease. |
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