Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus

Wanfeng Xie·Guanghong Liang·Aizhen Huang·Feiping Zhang·Wenshuo Guo

Abstract Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expression profile of P. massoniana infested by Bursaphelenchus xylophilus.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistancelike protein,and the gene encoding a putative nematode resistance protein.Increased infestation pressure(number of nematodes inoculated)caused a continuous decline in the gene expression of stem samples.An infestation of P.massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.A downregulation of auxin responsive family protein was observed in infested samples,which resulted in a suppression of plant growth.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and growth regulation was observed in P.massoniana,together with a decrease in the levels of phytoalexinlike secondary substances,all of which resulted in withering and ultimately death of P.massoniana.

Keywords Auxin/IAA·Bursaphelenchus xylophilus·Pinus massoniana·Resistance gene·Phytoalexin

Introduction

Masson pine(Pinus massoniana)is the most widely distributed and abundant pine species in China.Pine wood nematode(Bursaphelenchus xylophilus;PWN)is the main causative pathogen of a destructive disease that endangers pine growth.Nematode infestation decreases the transpiration and turpentine secretion of pines,thereby resulting in withering and eventual death(Fukuda 1997).Pine wilt disease(PWD)was first reported in Jiangsu Province,China in 1982.Over the next 25 years,PWD spread across 110,000 ha in China,leading to the death of ＞50 million stems of pine trees and significant losses to the national forestry economy(Zhao 2008).PWD is thus one of the main risk factors in P.massoniana production.

Nematode infestation usually causes the death of parenchyma cells,which is ascribed to the dysfunction of the water-conducting system,thus leading to the quick death of pine trees(Futai 2013).Other symptoms detected during the progress of nematode infection include reduction of photosynthesis, accumulation of phytotoxic compounds and ethylene,destruction of cambium,and denaturation of xylem(Fukuda 1997).

When a plant is afflicted by disease,it changes its gene expression profile,thereby influencing its disease defense capability.Xu et al.(2013)used suppression subtractive hybridization technique to construct a differential gene expression library of P.massoniana with B.xylophilus infestation.The results showed that these genes involved in signal transduction, transcription, translation, and secondary metabolism were upregulated in P.massoniana at 24 h and 72 h after B.xylophilus infestation,and the expression of other classes of genes associated with stress response increased at 72 h after infestation.P.massoniana with different resistance reactions had varying degrees of molecular responses after B.xylophilus infestation.Zheng et al.(2015)compared the different cultivars of P.massoniana with B.xylophilus infestations and showed that expression of copper/zinc superoxide dismutase(Cu-Zn SOD),glutathione S-transferases,and ascorbate peroxidase,as well as photosynthesis-related ATP synthase in the resistant cultivar,was significantly higher than that in the susceptible cultivar, suggesting that these differences improved disease resistance in P.massoniana.Hirao et al.(2012)documented that high transcript levels of pathogenesis-related genes and antimicrobial-related genes were rapidly induced in susceptible trees,whereas a moderate defense response was demonstrated in resistant trees against PWN infection.These preliminary results revealed the molecular responses of P. massoniana against B.xylophilus infestation.

With the continuous development of high-throughput sequencing technologies, methods in determining the transcription levels of various genes in response to different environmental conditions have been developed.Gao et al.(2013)used a high-throughput RNA sequencing(RNA-seq)technology to study the transcriptome dynamic expression of anti-disease transgenic potato and its nontransgenic wild-type potato infested by Phytophtora infestans.Of the 29,319 detected gene transcripts,2531 genes were differentially expressed in at least one test sample compared to the other samples.Westermann et al.(2012)introduced an RNA-seq technology for the simultaneous analysis of gene expression between host and pathogens, thereby facilitating the elucidation of the interaction-related mechanism between host and pathogens.These studies used the RNA-seq technology to provide a reference for transcript responses of forest trees to pathogen infection.

The transcription response of P.massoniana with B.xylophilus infestation is also an important basis for the downstream defense system of P.massoniana.Understanding this process helps to establish the actual mechanism underlying the physiological and biochemical changes occurring in P.massoniana upon B.xylophilus infestation.This study used RNA-seq technology to identify the dynamically expressed transcripts of P.massoniana at various time points of B.xylophilus infestation and detect changes in the expression of genes related to major metabolic pathways.

Materials and methods

Plant materials

The tested P.massoniana trees were 2 years old and were growing in the greenhouse of the Institute of Forest Protection at Fujian Agriculture and Forestry University,China.B.xylophilus that exhibited strong virulence was isolated from diseased trees of P.massoniana.B.xylophilus was grown in Pestalotiopsis colonies for pure culture and stored in a 4°C refrigerator for subsequent use.

Infection of P.massoniana by B.xylophilus

P.massoniana seedlings of approximately 40 cm in height that were grown under normal growth conditions were subjected to inoculation with B.xylophilus.The amount of B.xylophilus used for inoculation was 2000 nematodes for each seedling.Inoculation of sterile water was used as a control.These seedlings inoculated with nematodes or sterile water were all kept for 1,2,and 3 days respectively.Each treatment or control group included 3 replicates.On days 1,2,and 3 of inoculation,pine needles were collected from treatment and control groups and rapidly frozen in liquid nitrogen for RNA extraction.

Extraction of mRNA from needle tissues of P.massoniana and construction of library

An E.Z.N.A.®Plant RNA Kit(Omega Bio-tek Inc.,USA)was used for the extraction of RNA from the host plant to be tested. The operation procedure was performed according to the instruction manual of the reagent kit.RNA integrity was measured on an Agilent 2100 Bioanalyzer system(Agilent Technologies,USA)for quality control.Total RNA from the needles of seedlings inoculated with sterile water for 1 d,2 d,and 3 d,were mixed equally and taken as the control for sequencing.The mRNA from the total RNA of treatment and control was respectively enriched by Dynabeads Oligo(dT)25beads(Life Technologies Co.,US).It was then fragmented into small fragments by using fragmentation buffer(New England Biolabs Ltd.,UK).These mRNA fragments were reversely transcribed into cDNA with random primers.After that,second-strand cDNA was synthesized and purified with QIAquick PCR extraction Kit(QIAGEN Inc.,USA).The cohesive ends of dscDNA were filled into the blunt ends and then added with poly(A),and ligated to Illumina sequencing adapters.The ligation products were size selected by agarose gel electrophoresis and amplified by PCR. These cDNA libraries were sequenced using Illumina HiseqTM2500 by Gene Denovo Biotechnology Co.,China.

Bioinformatics analysis of the transcriptomic data from P.massoniana

The outcomes of the sequencing usually included adaptorcontaining reads and reads with more than 50%low-quality bases (Q-value ≤10) or more than 10% unknown nucleotides.These reads were removed and the rest of the reads were retained as target reads.Transcriptome de novo assembly was carried out using the Trinity method to obtain the full length of each mRNA(Grabherr et al.2011).The number of the reads that uniquely mapped to the assembled sequence were regarded as the expression abundance of unigene.The unigene expression was calculated and normalized to Reads Per kb per million reads(RPKM).The formula is as follows,RPKM=(1,000,000×C)/(N×L/1000).Here,C is the number of the reads that are uniquely mapped to the unigene A,N is the total reads that are uniquely mapped to all unigenes,and L is the length(base number)of unigene A(Mortazavi et al.2008).

To annotate the unigenes,the BLASTx program(http://www.ncbi.nlm.nih.gov/BLAST/)with E-value threshold of 1E-5 to NCBI non-redundant protein (Nr) database(http://www.ncbi.nlm.nih.gov)and the Swiss-Prot protein database(http://www.uniprot.org/)were selected.Protein functional annotations were obtained according to the best alignment results.

Analysis of differentially expressed genes

To identify differentially expressed genes(DEGs)across samples or groups,the edgeR package(http://www.r-pro ject.org/)was used.Genes with a fold change ≥2 and a false discovery rate(FDR)＜0.05 in a comparison between treatment and control groups were regarded as significant DEGs.DEGs were then subjected to enrichment analysis of GO functions and KEGG pathway.

As an international standardized gene functional classification system, GO was implemented for functional annotation of the unigenes, and the classification was obtained from Nr annotation results. GO has three ontologies:molecular function,cellular component,and biological process.The basic unit of GO is GO-term.Each GO-term belongs to a type of ontology.GO enrichment analysis yields all GO terms that were significantly enriched in DEGs in comparison to the genome background,and filters the DEGs that correspond to biological functions.First,all DEGs were mapped to GO terms in the Gene Ontology database(http://www.geneontology.org/),gene numbers were calculated for every term,significantly enriched GO terms in DEGs comparing to the genome background were defined by a hypergeometric test.The calculating formula of P-values is as follow,

where N is the number of all genes with GO annotation;n is the number of DEGs in N;M is the number of all genes that are annotated to the certain GO terms;m is the number of DEGs in M.The calculated p value was run through FDR correction,taking FDR ≤0.05 as a threshold.GO terms meeting this condition were defined as significantly enriched GO terms in DEGs.This analysis was able to recognize the main biological functions that DEGs exercise.

Pathway enrichment analysis

Genes usually interact with each other to play roles in certain biological functions.Pathway-based analysis helps to further understand gene biological functions.KEGG is the major public pathway-related database(Kanehisa et al.2008).Pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways in DEGs compared with the whole genome background.The calculating formula is the same as that in GO analysis.Here N is the number of all genes with KEGG annotation,n is the number of DEGs in N,M is the number of all genes annotated to specific pathways,and m is a number of DEGs in M.The calculated p value was run through FDR Correction,taking FDR ≤0.05 as a threshold.Pathways meeting this condition were defined as significantly enriched pathways in DEGs.

qPCR analyses to determine the gene dynamic expression

To validate the gene expression abundance detected from RNA-seq,quantitative polymerase chain reaction(qPCR)was used to determine the relative mRNA expression levels of the genes in P.massoniana upon B.xylophilus infestation compared with the control P.massoniana(samples inoculation with sterlize ddH2O for the same number of days). Healthy P. massoniana seedlings were again subjected to inoculation with B.xylophilus,both treatment group and the corresponding control group were sampled respectively after 1,2,and 3 d of inoculation.The treatment and control groups both included 3 biological replicates.The total RNA from each tested P.massoniana sample was reversely transcribed into cDNA by using the TIANScript RT Kit(TIANGEN Biotech(Beijing)Co.,Ltd.).A TransStart Green qPCR SuperMix Kit(Trans-Bionovo Co.,Ltd.)was used for the qPCR amplification.The reaction was performed in the QuantStudioTM6 Flex Real-Time PCR System(ABI,Thermo Fisher Scientific Inc.,USA),and threshold cycle values(Ct)were recorded for each candidate mRNA in both the control and test samples.The method of 2-ΔΔCtwas used to analyze the relative folds of gene change expression(Livak and Schmittgen 2001)and the gene of actin(Unigene 0040800),which was obtained from RNA-seq,was taken as a reference gene to normalize the expression of candidate genes in the tested sample.

Detection of the contents of total phenols and flavonoids

Pine needles of P.massoniana in the treatment with 3,5,7,9,11,and 14 days of inoculation and corresponding control groups for the same numbers of days,were collected to detect the contents of total phenols and total flavonoids.Detection was conducted following the manual of the relevant Kit(Nanjing SenBeiJia Biological Technology Co.,Ltd.).The treatment and control groups both included three biological replicates.

Detection of the gene expression on P.massoniana infected with different quantities of B.xylophilus

P.massoniana seedlings were inoculated with 500,1000,2500,5000,10,000,and 20,000 nematodes of B.xylophilus for 2 days,another group with sterilized water inoculation was taken as corresponding control.The tissues of pine stem and needle leaves were collected and then frozen in liquid nitrogen for RNA extraction.The total RNA was reversely transcribed into cDNA.Relative mRNA expression levels of disease resistance gene, putative cyst nematode resistance protein,and auxin-responsive family protein genes in treatment and control groups were assessed by using qPCR.

Results

Numbers of differentially expressed genes in infested and control pine needles at various durations after infestation

After a standard analysis and quantity control of the outcome of RNA-seq on the individual test sample(Fig.S1,S2;Table S1),statistical analysis of the expression level of gene sequencing data from different samples was conducted,in order to compare the DEGs at different days after infestation.Compared to the control,infested seedlings for 1 day had 1760 DEGs, which included 674 upregulated genes and 1086 downregulated genes(Fig.1A,D);after 2 days of infestation there were 1806 DEGs,which included 872 upregulated genes and 934 downregulated genes(Fig.1B,D);and after 3 days of infestation there were 742 upregulated genes and 983 downregulated genes(Fig.1C,D).Compared to the controls,after 1 and 2 days of infestation infected trees had 633 DEGs in common;after 1 and 3 days of infestation trees had 741 DEGs in common;and after 2 and 3 days of infestation trees had 619 DEGs in common.Compared to the control sample,after 1,2,and 3 days of infestation infected trees had 387 DEGs in common(Fig.1E,S3A).

GO functional enrichment analysis and significance analysis of DEGs

Compared to the control,the genes differentially expressed in P.massoniana after 1 day of infestation were associated with 18 biological processes(e.g.,metabolic processes,cellular processes, and development processes). The expression of genes associated with viral replication and immune system processes was downregulated,whereas that of growth-related genes was upregulated.In addition,genes related to the extracellular regions of cellular components were upregulated.In terms of molecular functions,genes related to cells and organelles were differentially expressed;those associated with electron-carrier capacity,auxiliary transport protein activity,molecular transducer activity,and enzyme regulator activity were downregulated,whereas those related to six functions,viz.catalytic ability,binding,transporter activity,structural molecule activity, translation regulator activity, and antioxidant activity,were differentially expressed(Fig.S4A).

Fig.1 Number of DEGs and Venn diagram of the needles of P.massoniana after different days of B.xylophilus infestation.The genes expression level from the P.massoniana infested with B.xylophilus after 1,2,and 3 days compared to that of the control group respectively.Genes with a fold change ≥2 and at false discovery rate(FDR)＜0.05 in a comparison as significant DEGs.TR1 versus CT,the DEGs from the P.massoniana after 1d inoculation compared to the control.TR2 versus CT,the DEGs from the P.massoniana after 2d inoculation compared to the corresponding control.TR3 versus CT,the DEGs from the P.massoniana after 3d inoculation compared to the corresponding control.The DEGs are presented as scatter plot shown in A-C,which represent TR1 versus CT,TR2 versus CT,and TR3 versus CT,respectively.The red scatters represent up-regulated genes,and the green scatters represent down-regulated genes,whereas the blue scatters represent not DEGs.The total number of upregulated and down-regulated genes from P.massoniana infected with B.xylophilus after 1d,2d,and 3d compared to that of the control group was shown in(D).E is a Venn diagram analysis on the DEGs overlap in among the P.massoniana infected with B.xylophilus after 1d,2d,and 3d compared to that of the control group,respectively

In terms of functional classification of biological processes after 2 days of infestation,the genes associated with cell death and rhythmic process were upregulated,whereas those related to viral reproduction were downregulated.The number of downregulated genes associated with the biological processes such as localization, multicellular organismal process,developmental process,reproductive process, cellular component organization, and cellular component biogenesis was significantly higher than that of upregulated genes.In terms of molecular function,the genes associated with auxiliary transport protein activity and enzyme regulator activity were downregulated. In addition,the number of downregulated genes that were related to the membrane-enclosed lumen and the extracellular region was significantly higher than that of upregulated genes(Fig.S4B).

In terms of the molecular functions at 3 days after infestation,the genes associated with auxiliary transport protein activity, enzyme regulator activity as well as structural molecule activity,and immune system processes were downregulated.The number of downregulated genes associating with processes of multi-organisms and cellular components organization was significantly higher than that of upregulated genes.The number of downregulated genes associated with cellular components (e.g., organelle,organelle part, extracellular region, and membraneenclosed lumen)and antioxidant activity were both higher than that of upregulated genes.In contrast,growth-related and extracellular region part genes in the cellular component GO category were upregulated(Fig.S4C).These findings indicated that nematode infestation resulted in the downregulation of genes that play major functions in the needles of P.massoniana and that this might have affected its resistance.

Significance and enrichment analyses of DEGs involved in the metabolic pathways

Significance and enrichment analyses of DEGs in the pine needles after 1 day of infestation were annotated in 79 metabolic pathways.Among the 79 pathways,DEGs in 12 pathways were significantly enriched, which included phenylalanine metabolism,secondary metabolites biosynthesis,phenylpropanoid biosynthesis,metabolic pathways,and plant hormone signal transduction.DEGs in needles at 2 days after infestation had annotations in 76 pathways,of which 9 pathways were significantly enriched, e.g.,phenylalanine metabolism,secondary metabolites biosynthesis,phenylpropanoid biosynthesis,metabolic pathways,and flavonoid biosynthesis.DEGs in the needles at 3 days after infestation had annotations in various pathways,including phenylalanine metabolism,secondary metabolites biosynthesis,phenylpropanoid biosynthesis,thiamine metabolism,and diterpenoid biosynthesis(Table 1).

Further enrichment analysis of DEGs simultaneously found in the needles at different time points after infestation and in control needles showed significant enrichment in the categories of secondary metabolite biosynthesis and phenylpropanoid biosynthesis(Table 2),suggesting that nematode infestation affected the secondary metabolites of the needles.These substances are the major substances of chemical defense in plants.

Changes in the expression of secondary metabolismrelated genes after infestation

Results of RNA-seq showed that dihydroflavonol-4-reductase (DFR) and cinnamyl alcohol dehydrogenase(CAD), which are associated with the biosynthesis of anthocyanins and lignin,respectively,were downregulated after infestation relative to that observed in the controls(Fig.S3B).Further analysis of the expression levels of these two genes by using quantitative PCR(qPCR)showed that DEF and CAD were downregulated after 1,2,and3 days of infestation compared to that in the corresponding controls (Fig.2), suggesting that nematode infestation inhibited the biosynthesis of secondary metabolites such as various classes of phytoalexins.

Table 1 KEGG enrichment analysis of differentially expressed genes in the needles of P.massoniana after different days of B.xylophilus infestation

Table 2 KEGG enrichment analysis of the common differentially expressed genes in the needles of P.massoniana after different days of B.xylophilus infestation

Further determination of total flavonoid and total phenolic contents of P. massoniana with and without B.xylophilus infestation at different time points showed that the total flavonoid content after 3 days of infestation was significantly lower than that of the controls at the corresponding time point.The highest total flavonoid content was recorded at 7 days after infestation and did not differ among groups.The total flavonoids content at 9 days after infestation showed a gradual decrease,whereas that after 14 days was significantly lower.Similarly,total phenolic content at different time points after infestation initially increased and then later decreased.The total phenolics content at two weeks after infestation was significantly lower than that of the controls at the corresponding time point(Fig.3).

Changes in the expression of genes associated with signal transduction of plant-pathogen interactions

RNA-seq analysis showed that among the plant-pathogen interactions of P.massoniana after B.xylophilus infestation, the coiled-coil-nucleotide-binding site-leucine-rich repeat(CC-NBS-LRR)resistance protein,toll-interleukin-1 receptor/nucleotide-binding site(TIR/NBS),and NBS/LRR genes related to hypersensitive response processes were downregulated.In addition,a plant disease resistance gene(R gene)with a high degree of homology to LRR from Pinus sylvestris was also downregulated in control P.massoniana.qPCR of genes after 1,2,and 3 days of infestation relative to that in the control identified downregulated genes,and these results were in agreement with those of RNA-seq analysis(Fig.4,S3B).

Changes in the expression of defense-,growthand development-relevant genes

Fig.2 Changes of DFR and CAD genes expression in P.massoniana after different days of B. xylophilus infestation. Differentially expressed genes obtained from RNA-seq were validated by qPCR.Actin was used as a reference,and the expression level of each gene in P.massoniana after 1,2,and 3 days of B.xylophilus infestation was then compared with those in P.massoniana with sterilized water inoculation under same time point,of which all the gene were set at 1.0.Error bars indicate the standard deviations of four biological replicates.DFR,dihydroflavonol-4-reductase,CAD,cinnamyl alcohol dehydrogenase

Fig.3 Changes of total flavonoids and total phenolics content in P.massoniana after different days of B. xylophilus infestation. P.massoniana infested with B.xylophilus was taken as treatment,while the sample infested with sterlize ddH2O was regarded as control.After 3,5,7,9,11,and 14 days,B.xylophilus infested pine needles and the control was sampled for the detection of total flavonoids and total phenolics content.For each pair of columns,an asterisk(*)indicates statistical groups that are significantly different(p ＜0.05),and double asterisks represent highly significant differences(p ＜0.01) while triple asterisks represent extremely remarkable difference(p ＜0.001)by using Tukey's range test.FW,fresh weight

Fig.4 Changes in gene expression associated with the signal transduction of the pathogen in P.massoniana after different days of B.xylophilus infestation.Actin was used as a reference,and the expression level of each gene in P.massoniana after 1,2,and 3 days of B.xylophilus infestation was then compared with those in P.massoniana with sterilized water inoculation under same time point,of which all the gene were set at 1.0.Error bars indicate the standard deviations of four replicates

By comparing the RNA-seq results between P.massoniana with different days of B.xylophilus infestation,we determined that the gene expression of putative cyst nematode resistance protein(CNR)and late embryogenesis abundant-like protein(LEA)was significantly lower than that in the control plant(Fig.S3B).The dynamic expression of these two genes as indicated in the qPCR analysis was in agreement with the RNA-seq findings(Fig.5).

Infestation also affected the expression of some growth and development-related genes in P. massoniana. For example,gene of auxin-responsive family protein(ARF)was downregulated after infestation, which further decreased over time(Fig.S3B).In contrast,the auxin-repressed protein was up-regulated after infestation(Fig.5).

Changes in gene expression in various tissues after different quantities of B.xylophilus infestation

To further reveal the relationship between the target gene expression and quantities of infestation,we used different inoculation quantities of nematodes, after which the expression of CNR,R gene,and ARF genes was examined.Different quantities of infestation resulted in the downregulation of the R gene in the stems and needle leaves of P.massoniana compared to that observed in the control plants.When the amount of nematodes used in inoculation was 500,the expression of the ARF was higher than that in the control,whereas that of ARF continuously declined and was lower than of the control, under the inoculation amounts ≥1000.Varying quantities of infestation resulted in the downregulation of the CNR gene in the stems.With inoculation using increasing amounts(≥1000)of nematodes, the expression of the CNR gene continuously decreased.The gene expression was also down-regulated in the needle leaves after nematode inoculation,but it was upregulated when the nematode inoculation quantity was≥5000(Fig.6A,B).This results suggested that the R gene obtained from RNA-seq was correlated with disease resistance in P.massoniana.

Fig.5 Changes of defense-relative and auxin-responsive genes expression in P.massoniana after different days of B.xylophilus infestation.Actin was used as a reference,and the expression level of each gene in P.massoniana after 1,2,and 3 days of B.xylophilus infestation was then compared with those in P.massoniana with sterilized water inoculation under same time point,of which all the gene were set at 1.0.Error bars indicate the standard deviations of four replicates

Fig.6 Changes in gene expression in the stems and needle leaves of P.massoniana after different quantities of B.xylophilus infestation.A total of 500,1000,2500,5000,10,000,and 20,000 pieces number of nematode was respectively inoculated on the P.massoniana for 2 days.The expression levels of R gene,ARF,peroxidase,and CNR in P.massoniana with B.xylophilus infestation were then compared with those in P.massoniana with sterilized water inoculation,of which all the gene expression level were set at 1.0.Actin was used as a reference. Error bars indicate the standard deviations of four replicates.A the relative mRNA expression level in the stems,B the relative mRNA expression level in the needles

Discussion

The gene expression pattern of infested P.massoniana shows the immediate response of the host plant to the disease,thus the first three days after infestation were a period during which no phenotypic response to infestation was recorded in spite of notable changes in gene expression.

Plants must identify pathogens to form defense responses.B.xylophilus infestation inhibited the gene expression of some specific recognition factors in P.massoniana such as the CC-NBS-LRR resistance protein and R genes.NBS and LRR domains of the CC-NBS-LRR resistance proteins are the two most important domains in the ubiquitous disease resistance genes(Dangl and Jones 2001).NBS contains a highly conserved P-loop,kinase-2,and Gly-Leu-Pro-Leu motifs and functions in signal transmission(Tan and Wu 2012).LRR has a high-affinity domain and provides an effective locus for protein-protein interactions(Jones and Dangl 2006;Ellis et al.2000).With increasing quantities of B.xylophilus infesting P.massoniana,the degree of R gene (Unigene 0041321) downregulation intensified.This gene encodes a protein that also contains an LRR domain and is highly homologous to an R gene of P. sylvestris. These R gene encoded proteins play an important role in disease resistance and defense in plants and are important factors in plant immunity.Increased expression of the R gene enhances disease resistance in plants(Li and Asiegbu 2004).Therefore,the downregulated expression of R gene and NBS/LRR in P.massoniana after B.xylophilus infestation hindered the identification and signaling of pathogenic factors and affected the responses of the defense system in plants.

Inhibition of signal transduction and gene expression changed the expression of the R gene in infected P.massoniana,thereby suppressing the expression of the CNR gene in the stems and needles of the seedlings.With increasing numbers of nematodes, such inhibition was enhanced.The defense capability of P.massoniana against B.xylophilus was associated with the level of expression of the R gene.McLean et al.(2007)conducted molecular cloning overexpression of a nematode resistance gene in soybean,Hs1pro-1,which was initially identified in beet(Cai et al.1997).Nematode resistance in T1 transgenic soybeans increased by 70%,compared to that in nematodesensitive wild-type control soybeans,demonstrating that these types of nematode resistance genes improved disease resistance against nematode infestation in soybeans.

The LEA gene in needles of P.massoniana after B.xylophilus infestation was downregulated.The accumulation of LEA proteins in plant cells is caused by biotic and abiotic stress-induced dehydration(Hanin et al.2011;Liu et al.2013).Changes in the expression of LEA in the needle of P.massoniana after B.xylophilus infestation might be associated with physiological changes, including the gradual dehydration and withering of the infested stems.The downregulated expression of these defense-related genes in P.massoniana infested with B.xylophilus was detrimental to the development of a plant defense system and in turn caused a decline in the physiological defense capabilities of the plant.

From a physiological and defense perspective, the biosynthesis of phytoalexin secondary metabolites in P.massoniana after B.xylophilus infestation was significantly affected.The expression of DFR and CAD involved in the two phenylpropanoid metabolic pathways,were downregulated.DFR catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins(two flavonoid classes that are important to plant survival(Petit et al.2007)),and also one kind of phytoalexin that has antioxidant properties that enhance disease resistance.Overexpression of DFR in the plant results in an increase in the anthocyanin content and the development of disease resistance(Kortekamp 2006).CAD catalyzes the last step in the biosynthesis of the monolignols,which is a lignin precursor.The deposition of lignin in the plant cell wall during pathogen invasion is thought to be a method of plant defense,and the upregulation of CAD also leads to enhancement of disease resistance(Tronchet et al.2010).In the present study,DFR and CAD were downregulated in B.xylophilus-infected P.massoniana,which is in agreement with the observed decrease in the level of total flavonoids.The decrease in the total flavonoid content upon infestation indicated the rapid response of the tested susceptible line,as reported earlier by Hirao et al.(2012).Total phenolics declined significantly after two weeks of infestation.

Due to the failure of pathogen defense in tissues,the growth and development of Pinus would be inhibited,caused mainly by the downregulation of ARF,as well as the upregulation of auxin-repressed protein in the needle.ARF can be upregulated by auxin,the plant hormone that is key to the regulation of plant growth and development.The gene encoding the auxin-repressed protein,which exhibits a reverse response to auxin as the typical auxin-responsive family protein,is negatively associated with hypocotyl elongation(Park and Han 2003).B.xylophilus infestation of Pinus resulted in changes in the expression two genes that negatively affected the plant.Our previous studies showed that B.xylophilus infection influenced the expression of miRNAs related to plant hormone signal transduction in P. massoniana, decreasing plant hormone synthesis and ultimately affecting the growth of Masson pine(Xie et al.2017).

Fig.7 Schematic summary of a model of molecular response in P.massoniana to B.xylophilus infestation.The figure indicates that the infestation of B.xylophilus causes the down-regulation of genes involved in disease-resistance and system-defense,which leads to decrease in the levels of phytoalexin-like secondary substances,all of which resulted in withering and ultimately the death of P.massoniana.A detailed explanation is presented in the text.Solid arrows point to the brief gene regulation network in P.massoniana,based on the results of our study

In conclusion,the expression of the pathogenic-signaling-related genes NBS/LRR and R gene was inhibited in P.massoniana after B.xylophilus infestation,followed by a downregulated expression of the resistance-associated genes CNR and LEA,resulting in declining expression of the biosynthesis-related genes CAD and DFR.The phenols and flavonoid contents changed in the infested P.massoniana,and the overall defense capability of the plant was reduced.As a result,expression of cell growth and development-related genes decreased,plant growth was inhibited,plant cells aged at accelerated rates,and the seedlings eventually withered and died(Fig.7).