- Open Access
De novo transcriptomic assembly and mRNA expression patterns of Botryosphaeria dothidea infection with mycoviruses chrysovirus 1 (BdCV1) and partitivirus 1 (BdPV1)
- Lihua Wang†1, 2, 3,
- Hui Luo†1, 2, 3,
- Wangcheng Hu1, 2, 3,
- Yuekun Yang1, 2, 3,
- Ni Hong1, 2, 3,
- Guoping Wang1, 2, 3,
- Aiming Wang4 and
- Liping Wang1, 2, 3Email author
© The Author(s). 2018
- Received: 8 February 2018
- Accepted: 27 July 2018
- Published: 13 August 2018
Pear ring rot, caused by Botryosphaeria species, is responsible for substantial economic losses by causing severe recession of pear tree growth in China. Mycovirus-mediated hypovirulence in plant pathogenic fungi is a crucial biological method to control fungal diseases.
We conducted a large-scale and comprehensive transcriptome analysis to identify mRNA in B. dothidea in response to mycovirus. De novo sequencing technology from four constructed libraries of LW-C (Botryosphaeria dothidea chrysovirus 1, BdCV1), LW-P (Botryosphaeria dothidea partitivirus 1, BdPV1), LW-CP (LW-1 strain infection with BdCV1 and BdPV1), and Mock (free virus) was used to investigate and compare gene expression changes in B.dothidea strains infected with mycovirus.
In total, 30,058 Unigenes with an average length of 2128 bp were obtained from 4 libraries of B. dothidea strains. These were annotated to specify their classified function. We demonstrate that mRNAs of B. dothidea strains in response to mycovirus are differentially expressed. In total, 5598 genes were up-regulated and 3298 were down-regulated in the LW-CP group, 4468 were up-regulated and 4291 down-regulated in the LW-C group, and 2590 were up-regulated and 2325 down-regulated in the LW-P group. RT-qPCR was used to validate the expression of 9 selected genes. The B. dothidea transcriptome was more affected by BdCV1 infection than BdPV1. We conducted GO enrichment analysis to characterize gene functions regulated by B. dothidea with mycovirus infection. These involved metabolic process, cellular process, catalytic activity, transporter activity, signaling, and other biological pathways. KEGG function analysis demonstrated that the enriched differentially expressed genes are involved in metabolism, transcription, signal transduction, and ABC transport. mRNA is therefore involved in the interaction between fungi and mycovirus. In addition, changes in differential accumulation levels of cp and RdRp of BdCV1 and BdPV1 in B. dothidea strains were evaluated, revealing that the accumulation of BdCV1 and BdPV1 is related to the phenotype and virulence of B. dothidea strain LW-1.
The identification and analysis of mRNAs from B. dothidea was first reported at the transcriptome level. Our analysis provides further insight into the interaction of B. dothidea strains infection with chrysovirus 1 (BdCV1) and partitivirus 1 (BdPV1) at the transcriptome level.
- Pear ring rot disease
- Botryosphaeria dothidea
- De novo transcriptional sequencing and analysis
- KEGG pathway
- GO enrichment analysis
- Differential expression genes (DEGs)
- Fungi-mycovirus interaction
Pear ring rot, caused by the destructive pathogen Botryosphaeria, is responsible for substantial economic losses through widespread fruit rots and stem canker. This caused a severe recession in the growth of pear fruit trees in China [1–5]. Due to the lack of disease-resistant varieties, many fungicides are recommended for treatment of Botryosphaeria canker, which induces drug resistance and environmental pollution . It is urgent to find new, safe, and effective means to control pear ring rot disease. Mycovirus-mediated hypovirulence in plant pathogenic fungi is a powerful method to control fungal crop diseases such as the hypovirulent strain of C. parasitica (CHV1) to heal cankers of chestnut blight, and Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) to control the rapeseed stem rot [6–13]. Recent studies of fruit tree fungal diseases have reported that the mycovirus Rosellinia necatrix megabirnavirus 1 (RnMRV1) has potential to control white root rot (Rosellinia necatrix) diseases of fruit trees . In recent years, several dsRNA mycoviruses have been identified and sequenced in B. dothidea strains isolated from pear trees with ring rot and stem wart symptoms in China [14, 15]. Chrysovirus BdCV1 and partitivirus BdPV1 mixed infection results in hypovirulent B. dothidea LW-1 strain in our study, revealing BdCV1 to be the first mycovirus found to be responsible for hypovirulence (reduced virulence), growth rate, and phenotypic sectorization of the phytopathogenic fungus B. dothidea . BdCV1 is therefore a good candidate for biological control of apple and pear ring spot diseases, while BdPV1 does not cause any visible symptoms in virulent pathogenicity [5, 16].
High throughput sequencing technology provided comprehensive information on gene expression. Measuring the expression patterns of mRNAs at the transcriptome level from fungi infection with mycovirus is vital to reveal the mechanism of mycovirus-mediated hypovirulence in pathogenic fungi [6, 17, 18]. Only a limited number of studies, using high throughput sequencing technologies and bioinformatics, have demonstrated transcriptional or translational changes in fungi infection with mycovirus, such as Cryphonectria parasitica, Aspergillus fumigatus, Neurospora crassa, Sclerotinia sclerotiorum, and Fusarium graminearum [17–24]. For instance, 150 genes representing a wide spectrum of biological functions were down-regulated in the strain Ep-1PN by Sclerotinia Sclerotiorum Debilitation-associated RNA Virus (SsDRV), of which S. sclerotiorum integrin like gene (SSITL) was verified affect pathogenic fungus virulence and growth rate [20, 23]. Genes associated with viral replication, maintenance of viral life cycle, transcription and translation machinery, and signal transduction were up-regulated while those including membrane-associated transporters and cellular transport systems were down-regulated based on a genome-wide transcriptome analysis of F. graminearum infected with Fusarium graminearum virus 1 (FgV1) [18, 21, 22, 25].
The main objective of our study is to characterize the mRNAs from B. dothidea involved in biological processes associated with the host infecting with mycovirus. Currently, there are no studies published on the B. dothidea transcriptome. No direct and detailed functional genomics resources in public databases for understanding the molecular mechanism of B. dothidea exist except the draft genome sequences from two B. dothidea strains reported, the pathogen of Apple ring rot disease and isolated from grapevine host, respectively [26, 27]. Therefore, large-scale transcriptome sequencing using Illumina sequencing technology was performed to explore the potential mRNAs expression in B. dothidea related to pathogenic factors and hypovirulence determinants in response to two mycoviruses BdCV1 and BdPV1.
Botryosphaeria dothidea strains and infection with BdCV1 and BdPV1
Three B. dothidea strains, LW-1, LW-C, and LW-P, infected with hypovirulent and non-hypovirulent mycovirus were generated [5, 16]. LW-1 (or designated as LW-CP) was infected with Botryosphaeria dothidea chrysovirus 1 (BdCV1) and Botryosphaeria dothidea partitivirus 1 (BdPV1), which was isolated and identified from the trunks of sand pear‘Jinshuiyihao’ cv., collected in Wuhan city, Hubei province of China . The LW-P strain infection with BdPV1 and LW-C with BdCV1 were derived from LW-1 by single hyphae isolation, respectively, as previously described [5, 16]. Meanwhile, mock derived from LW-C strain by protoplast isolation was virus-free as negative control. The fungal strains were grown at 25 °C in darkness on potato dextrose agar (PDA) medium and stored on PDA plate at − 4 °C or in glycerin at − 80 °C.
Total RNA extraction from B. dothidea strains
The mycelia of the mycovirus-infected three strains and the virus-free strain of B. dothidea were harvested on 5 day of PDA culture. The mycelia were frozen and ground using liquid nitrogen for nucleic acid extraction. Total RNAs were isolated and obtained with TRIzol Reagent (Invitrogen, USA), phenol and chloroform to remove proteins and ethanol precipitation according to manufacturer’s instructions. Total RNAs digested by DNase I (Takara, Dalian, China) were precipitated with ethanol and dissolved in DEPC treated water to be used as template for Next Genomics Sequencing (NGS). The RNA integrity Number (RIN > 7) and concentration of obtained tRNAs were further assessed using the Bioanalyzer Agilent 2100 and NanoDrop spectrophotometer (Agilent, USA). High quality samples were used to construct the four de novo libraries.
cDNA library construction and de novo sequencing
For each RNA-seq library, total RNA from three biological repetitions were pooled. Following the total RNA extraction and treatment with DNase I, mRNA was isolated and enriched by Oligo (dT). mRNA was cut into small fragments and cDNA synthesis was performed with random hexamer primers. PCR amplification was then prepared for library construction according to the Illumina RNA library protocol for transcriptome sequencing. Then, the quantification of the four cDNA libraries was verified by the Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System. Finally, the libraries were sequenced using the HiSeq 4000 instrument (Illumina, USA) at the Beijing Genomics Institute (BGI) (Shenzhen, Guangdong province, China).
Transcriptome data processing, de novo assembly, and functional annotation
Internal software was used to filter raw reads to generate clean reads data via the following processes: 1) The removal of adaptor-polluted reads, 2) removal of reads containing a high content (> 5%) of unknown bases (N), 3) removal of low-quality reads. Following filtering, the remaining reads were used for downstream analyses. Trinity was used to perform de novo assembly with clean reads which PCR duplication removed, and Tgicl was used to cluster transcripts to Unigenes. The sequence by Trinity was called transcripts. Then, gene family clustering with Tgicl for each sample’s Unigene to obtain final Unigenes was performed for downstream analyses. The Unigenes were divided into two classes, including clusters and singletons. The prefix used for clusters was CL with the cluster id behind it (in one cluster, there are several Unigenes with more than 70% similarity between them). The prefix used for singletons was Unigene. For unigene functional annotation analysis, NT (ftp://ftp.ncbi.nlm.nih.gov/blast/db), NR (ftp://ftp.ncbi.nlm.nih.gov/blast/db), GO (http://geneontology.org), COG (http://www.ncbi.nlm.nih.gov/COG), KEGG (http://www.genome.jp/kegg), SwissProt (http://ftp.ebi.ac.uk/pub/databases/swissprot), and InterPro (http://www.ebi.ac.uk/interpro) were used as functional databases. Blasts aligned unigenes to NT (NCBI non-redundant nucleotide sequences), NR (NCBI non-redundant protein sequences), COG (Cluster of Orthologous Groups), KEGG (Kyoto Encyclopedia of Genes and Genome), and SwissProt to obtain the annotation. Blast2GO with NR annotation to obtain the GO (Gene Ontology) annotation, and InterProScan5 was used to get the InterPro annotation [28–30]. The functional databases with a priority order of NR, SwissProt, KEGG, and COG were selected to determine the sequence annotation of unigenes. Coding sequences (CDS) were extracted from sequences of unigenes that best mapped to functional databases. ESTScan was used to predict coding regions of unigenes if no hits were obtained in any database mentioned above .
Differential gene expression analysis of mRNA in response to mycovirus
De novo sequencing from four constructed cDNA libraries designated as LW-CP, LW-C, LW-P, and Mock was performed. To analyze the differentially expressed genes (DEGs) between two samples in response to mycovirus, clean reads to assembled unigenes using Bowtie2 were mapped . Then, gene expression value was calculated with RSEM . DEGs were identified with PossionDis based on the Poisson distribution, which was performed as described by Audic et al. . The parameters of PossionDis was set as |Fold Change | > 2.0-fold (|log2FC| > 1) with a false discovery rate (FDR) < 0.001.
Clustering analysis of DEGs of B. dothidea strains infection with mycovirus
With DEGs, pheatmaps of hierarchical clustering were performed using the heatmap.2 package in R software . For clustering more than two groups, we used the intersecting DEGs.
GO and KEGG pathway analysis of DEGs
Gene Ontology (GO) classification and functional enrichment was performed on DEGs. GO displayed three ontologies including molecular function, cellular component, and biological process. In addition, KEGG pathway functional enrichment was also analyzed using a corrected P-value less than 0.05 for significantly enriched DEGs.
RT-qPCR analysis of gene mRNA expression level
RT-qPCR was used to validate DEG expression levels obtained from sequencing and to assess the expression levels of cp and RdRp from BdCV1 and BdPV1. Primers used are listed in Additional file 8. Total RNA was extracted from the four groups: Mock, LW-CP, LW-C, and LW-P cultured for 5 d, 10 d and 15 d using TRIzol (Invitrogen, USA) according to manufacturer’s instructions. Total RNA was digested by DNase I and used as template (Takara, Dalian, China). The cDNA was obtained to perform reverse transcription using PrimeScript™ RT reagent Kit with genomic DNA Eraser (Takara, Dalian, China). According to the manufacturer’s instruction, qPCR was performed using 2.5 μl diluted cDNA, 10 μl of the SYBR Premix Ex Taq II PCR mixture (Tli RNaseH Plus) (Takara, Dalian, China), 1 μl of each 5 mM primer, and deionized water to a final volume 20 μl. All reactions were run in triplicate. The qPCR was performed on the Bio-Rad iQTM5 Real-time System machine (BIO-RAD, USA). The products were verified by melt curve analysis. The 18 s gene from B.dothidea strains was used as the internal reference for standardization of cDNA expression levels from samples. mRNA relative expression level changes were quantified by a comparative CT method (ΔΔCT) using the formula 2-ΔΔCt .
Differential accumulation of cp and RdRp in B. dothidea infection with BdCV1 and BdPV1
De novo sequencing and unigene assembly of B. dothidea strains
Functional annotation of predicted proteins from B. dothidea unigenes
Summary statistics of the functional annotation of all unigenes from B.dothodea strains in public data bases
Number of unigenes
Annotated in at least one database
B.dothidea unigenes distribution in the top10 species
Macrophomina phaseoa MS6
Neofusicoccum parvum UCRNP2
Coniosporium apolis CBS 100218
Beauveria bassiana D1–5
Endocarpon pusillum Z07020
Pestalotiopsis fici W106–1
Glarea lozoyensis ATCC 20868
Nectria haematococca mpVI 77–13-4
Phaeosphaeria nodorum SN15
Setosphaeria turcica Et28A
Differential expression profiles of mRNAs from B. dothidea strains infection with mycovirus
Annotation of differentially expressed genes from B.dothidea strains in NR database
putative amino acid permease protein
Kelch repeat type 1
Major facilitator superfamily
GTP-binding domain HSR1-related protein
putative c6 transcription factor protein
CMP/dCMP deaminase zinc-binding protein
Glutathione S-transferase transferase
Putative ABC transporter protein
putative ubiquitin carboxyl-terminal hydrolase protein
Amino acid transporter transmembrane
Carboxylesterase type B
hypothetical protein MPH_13029
Mg2+ transporter protein CorA-like/Zinc transport protein, partial
Ctr copper transporter
Hypothetical protein CFIO01_01894
Glycoside hydrolase family 5
ATPase P-type K/Mg/Cd/Cu/Zn/Na/Ca/Na/H-transporter
hypothetical protein MPH_09082
Integral membrane protein
hypothetical protein MPH_01777
Fungal chitin synthase
Ferric reductase transmembrane component 4 precursors
Validation of mRNA transcriptome analysis data by RT-qPCR
qPCR analysis of de novo sequencing differentially expressed genes from B.dothidea across the three groups of LW-CP, LW-C, and LW-P
Aa Family ATPase
putative cytoskeleton organisation protein
HR1 repeat rho-binding protein
Hypothetical protein MPH_05438
Hypothetical protein MPH_07087
Putative ABC transporter
Effect of mycovirus infection on expression of predicted transcription factors
Differentially expressed TFs from B. dothidea strains belonging to 11 representative TF families were predicted in the three groups of LW-CP, LW-C, and LW-P, respectively
GO enrichment analysis of mycovirus-responsive unigenes from B. dothidea strains
In the LW-C group, 4879 DEGs were functionally assigned to: ‘metabolic process’ (GO: 0008152, 730 genes), followed by ‘cellular process’ (GO: 0009987, 541) in biological process. The ‘catalytic activity’ (GO: 0003824, 851 genes) in molecular function, were highly enriched. In the cellular component category, the ‘membrane’ (GO: 00016020, 238 genes), ‘integral component of membrane’ (GO: 00016021, 159 genes), and ‘intrinsic component of membrane’ (GO: 0031224, 238,159 genes) were significantly enriched (p-value is set as less than 0.05). In addition, other terms including ‘response to ‘stimulus’ annotated genes such as heat shock protein (Hsp)-related proteins, ‘Signaling’ and ‘transporter activity’ related to pathways involved in mycovirus-associated response were also enriched (Fig. 5b; Additional file 6: Table S6).
In the LW-P group, 2636 DEGs were functionally assigned. It is similar to the above two groups, such as ‘cell process’ (GO: 0009987, 271) in biological process, ‘catalytic activity’ (GO: 0003824, 453 genes) in molecular function, and ‘membrane’ (GO: 0016020, 131) in the cellular component category were highly enriched, respectively (Fig. 5c; Additional file 11: Figure S11).
KEGG pathway analysis of mycovirus-responsive genes from B. dothidea strains
From the above functional analysis, the DEGs associated with metabolic pathways, transport and catabolism, membrane transport, transcription, and signal transduction are presumed to play a role in the interaction of fungal defense and mycovirus counter defense strategies.
In this report, the transcriptome of B. dothidea strains was sequenced using the Illumina platform. A total of high-quality 18.87 Gb bases were obtained. We then assembled 4 fungi samples occurred in B. dothidea infection with mycovirus and obtained 30,058 Unigenes (≥200 bp). The N50 and GC content of unigenes was 3338 bp and 56.32%, respectively. We then annotated the unigenes using 7 functional databases. 24,836 (82.63%) unigenes were annotated with at least one functional database (Tables 1 and 2; Figs. 2 and 3). To the best of our knowledge, this is the first large-scale characterization of the B. dothidea genome at transcriptome level. Our results lay the foundation for further genomics research in Botryosphaeria species.
Previous research has demonstrated that mycovirus infections change the expression of a broad range of genes and cause hypovirulence or phenotypic alterations in the fungal host except plant [17–19, 22, 38, 39]. C. parasitica infected with Cryphonectria Hypovirus 1 (CHV1) is used as a model for studies on virus/host interaction. cDNA library analysis of differentially expressed genes involved viral replication, transmission, host growth, development, and defense. The pro1 and CpBir1 genes have important biological functions for hypovirus and chestnut blight fungus host [17, 40, 41]. In our study, RNA-Seq-based genome-wide expression profiling analysis in response to single or mixed mycovirus chrysovirus 1(BdCV1) and partitivirus 1 (BdPV1) infection was performed. The expression patterns of 9 putative genes involved in mycovirus stress measured by quantitative real-time PCR were consistent with their transcript changes as identified by RNA-seq (Table 4). It revealed specific and common alterations in host gene transcript accumulation following infection of B. dothidea by BdCV1 and BdPV1 . As expected, more transcriptional changes occurred in response to the hypovirulent LW-CP and LW-C compared to the LW-P (Table 3; Fig. 4), revealing that chrysovirus 1(BdCV1) would have a stronger effect than partitivirus 1 (BdPV1) on the B. dothidea transcriptome, and gene expression changes in transcriptome caused by hypovirulent and non-hypovirulent mycoviruses were related to the observed host phenotypes and pathogenicity [17, 18]. Meanwhile, changes in differential accumulation of BdCV1 and BdPV1 in B. dothidea strains were demonstrated that the accumulation level of BdPV1 decreased by co-infection with BdCV1 (Fig. 1). It revealed that the BdCV1 and hypovirulent determinants may inhibit the expression level of BdPV1, prediction that it is the main reason to induce to the phenotype and hypovirulence of B. dothidea LW-1 strain (Figs. 1 and 4), which need to be further verified.
It is known that transcription factors play important roles in fungi response to mycovirus. Mycovirus can activate TFs expression differentially . In this study, 1083 transcription factors classified into 18 families were predicted. The above data revealed many TFs from B. dothidea were expressed differentially among the mycoviruses, especially Zn2Cys6 and C2H2 zinc finger families (Table 5). This corroborates the results from the F. graminearum transcriptome, whose differentially expressed TFs included fungal-specific and dominant Zn2Cys6 and the C2H2 zinc finger members involved in transcriptional regulation [18, 22, 42, 43]. The results demonstrate that TFs are down-regulated in response to BdCV1 in LW-C, revealing that BdCV1 inhibits TFs expression in B. dothdiea (Table 5; Additional file 9: Table S9). In addition, a class of heat shock proteins (HSPs) was up-regulated in response to mycovirus infection (Additional file 6: Table S6), which supports the speculation that HSPs homologues are influenced by mycovirus .
To better survey the biological behavior of defense response, it is necessary to understand the functional distribution of these DEGs in B. dothidea following mycovirus infection based on the transcriptome and bioinformatics analysis [44–47]. Through the enriched GO terms analysis, it is also revealed that in biological processes, DEGs mainly distributed to metabolism. Differential expression of genes related to metabolism might be associated with the host phenotype [17, 39, 48]. In addition, GO terms for genes associated with signal transduction, including phosphatidylinositol signaling system and MAPK signaling pathway were more enriched in response to infection by BdCV1 compared to BdPV1. This reveals that it is necessary to determine the role of the MAPK signaling pathway in the regulation of mycovirus and host interactions . Genes involved in membrane, oxidoreductase activity, RNA biosynthetic processing, and ribosomal assembly were enriched in B. dothidea following mycovirus infection (Fig. 5; Additional file 11: Table S11), which indicates the diversity of genes affected by mycoviral infection. Furthermore, KEGG pathway analysis uncovered DEGs involved in important pathways. A mass of metabolism pathways, both primary and biosynthesis of secondary metabolism, are identified and significantly enriched in response to mycovirus infection (Fig. 6; Additional file 12: Table S12). Similar metabolic pattern is exhibited in different fungi-virus combinations, which indicated these pathways play important role in fungi host in response to mycoviral infection [17, 18, 22]. In addition, ‘carbohydrate metabolism’, ‘lipid metabolism’, ‘membrane transport system’, ‘transport and catabolism’, ‘translation’ and ‘signal transduction’ which were highly enriched (Fig.6; Additional file 12: Table S12). It also provides insight into the various biological pathways associated with mycoviral infection with plant pathogenic fungi.
RNAi machinery is involved in the regulation of fungi and mycovirus infection by controlling endogenous and exogenous RNA . Indeed, the biological functions of the RNA silencing pathway have been characterized in the Neurospora crassa, Cryphonectria parasitaica, Rosellinia necatrix, and F. graminearum [50–56]. As reported, FgAGO-1 and FgDICER-2 are responsible for hairpin RNA-triggered RNA silencing and related small interfering RNA accumulation in F. graminearum [18, 49, 51]. In this study, RNA interference components, including dicer-like (Dicer), argonaute-like (Ago), and RdRp genes in B. dothidea were expressed differentially in response to mycovirus infection (Additional file 5: Table S5). Small RNA sequencing demonstrates that microRNA exists and is expressed differentially in B. dothidea infection with mycovirus (not published). This suggests that B. dothidea possesses RNA silencing pathways for antiviral defense and endogenous gene regulation. These data indicate that mycovirus may activate host antiviral defense. Furthermore, it is necessary to determine and analyze the RNA silencing component responsible for transcriptional regulation and antiviral defense mechanism in B. dothidea.
In this study, 30,058 unigenes were obtained from hypovirulent and non-hypovirulent B. dothidea strains infected with mycovirus by de novo assembly. To identify potential mycovirus-responsive genes, DEGs were screened and further validated by RT-qPCR. Hierarchical clustering, which identifies gene sets of significantly differentially expressed genes occurred in B. dothidea infection with mycovirus, was performed. The expression analysis demonstrates that hypovirulent mycoviruse chrysovirus 1 (BdCV1) have a stronger effect than non-hypovirulent mycoviruse patittivirus 1 (BdPV1) on the B. dothidea transcriptome. This data indicates that the phenotypes and pathogenicity observed for mycovirus-infected B. dothidea are correlated with the numbers of DEGs and mycovirus variety [17, 18]. In addition, we found that most B. dothidea TFs were differentially expressed by each mycovirus, suggesting that fungal TFs have important roles in the response to mycovirus infection. Gene ontology (GO) enrichment and KEGG functional pathway analysis revealed that differential expression mRNAs played important roles in regulating the complex biological processes involved in B. dothidea infection with mycovirus. The obtained transcriptome data can provide molecular genomics resource for further functional characterization analysis of B. dothidea in response to mycovirus infection.
We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.
This work was supported by the National Natural Science Foundation of China [grant number 31471862], the Fundamental Research Funds for the Central Universities [grant number 2662016PY107] and the Earmarked Fund for Pear Modern Agro-industry Technology Research System [grant number CARS-28-15].
Availability of data and materials
The raw reads data from LW-CP, LW-C, LW-P and Mock samples have been submitted separately to the NCBI under the accession number SRP131718. The other data generated or analysed during this study are included in its additional files.
Wang LH, Luo H, Yang YK and Hu WC performed the experiments and analyzed the data. Wang GP, Hong N and Wang AM reviewed the manuscript. Wang LP designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
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