| Cistanche deserticola Ma,a rare and endangered plant,is the primitive plant recorded in ancient Chinese herbal medicine "Cistanche Herba".C.deserticola can obtain water and nutrients from its host plant and grow into Cistanche Herba only when it parasitizes on Haloxylon ammodendron or other plants.However,the parasitism of C.deserticola can stimulate the defense response of the host plant H.ammodendron,and the parasitism and anti-parasitism relationship are formed between C.deserticola and H.ammodendron,resulting in the low survival rate of C.deserticola,then in the decrease of the yield and profit of Cistanche Herba.Therefore,it is urgent to reveal the anti-parasitic defense response of H.ammodendron induced by C.deserticola,which will help solve the industrial bottleneck problem of low parasitic rate of Cistanche Herba.In this study,the effects of C.deserticola parasitic stress on the microstructure of H.ammodendron root cells were determined by paraffin section observation of the parasitized(NF)and normal growing roots of H.ammodendron(CK).Meanwhile,RNA-seq technique was used to compare the resistance of two groups:roots parasitized by C.deserticola(NF)and non-parasitized roots(P).H.ammodendron roots corresponding to vegetative growth stage(NF)and reproductive growth stage(F)of C.deserticola after successful parasitization.Then the main pathways involved in the regulation of parasitic stress were determined by comparing the transcriptional expression.On this basis,three homologous sequences of resistance genes were screened out,and their conserved structures were analyzed by bioinformatics to predict their biological functions.Finally,UPLC-MS/MS technology was used to detect the changes in the species and ion abundance of plant resistance related metabolites such as phenols and flavonoids in the parasitized roots(NF)and non-parasitized roots(P)of H.ammodendron.The changes of metabolites related to H.ammodendron resistance were searched and the molecular level regulation of resistance was analyzed from the perspective of metabolism.It provides experimental basis for the follow-up study on the stress resistance mechanism of H.ammodendron.The main results are as follows:1.Paraffin section staining showed that the proportion of central vessel cell area in the total cross section of H.ammodendron roots parasitized by C.deserticola was significantly reduced,while the thickness of vessel cell wall,central parenchyma cell group,root cortex and peripheral vessel cell were significantly greater than that of the normal growing H.ammodendron roots.It was speculated that H.ammodendron roots could store more nutrients by reducing vessel cell differentiation and increasing parenchyma cell number,and reduce water transfer to C.deserticola through vessel.At the same time,it strengthened its physical barrier by thickening the root bark and vessel cell wall to resist the invasion of C.deserticola haustorium and nutrition acquisition.2.Through transcriptome sequencing,differentially expressed genes with different parasitism results and periods after parasitism were identified.Compared with the normal growth of H.ammodendron(CK),differential genes were enriched in lignin biosynthetic process,plant-type secondary cell wall biogenesis etc.after H.ammodendron was parasitized,and accompanied by KEGG enrichment results of ferulic acid-5-hydroxylase gene(F5H)in phenylpropanoid biosynthesis significantly up-regulated,and coniferyl alcohol glucose transferase gene significantly down-regulated.It is speculated that the parasitized H.ammodendron roots mediate the could resistance reaction through the change of cell wall structure.At the same time,the differential genes in the unparasitized H.ammodendron roots were enriched in GO items such as inducing system resistance,system acquired resistance and salicylic acid mediated signaling pathway,with PRRs genes EIX1/2,XA21,CERK1 and LRR,receptor-like serine/threonine protein kinases EFR and FLS2 were significantly up-regulated in plant-pathogen interaction pathway,suggesting that unparasitized H.ammodendrum roots play a resistant response to parasitized signals.In addition,phytoalexin biosynthetic process and phytoalexin metabolic process were significantly enriched in H.ammodendron roots corresponding to the reproductive growth stage of C.deserticola,and the expression of salicylic acid,jasmonic acid,brassinolide and ethylene signal transduction genes TGA,JAR1,COI1,BIN2 and EIN2 were up-regulated in the plant hormone signal transduction pathway.It is speculated that the accumulation of defense compounds and the regulation of plant hormones could be involved in the regulation of resistance mechanism of H.ammodendron.3.Three potential resistance genes were screened from transcriptomic sequencing results,named RGA2-like,RGA3-like and RGA4-like,respectively.Bioinformatics analysis of their coding proteins showed that RGA2-like and RAG3-like proteins had NB-ARC domain.It has the highest homology with Beta Vulgaris resistance protein in phylogenetic tree.Ten conservative motifs were screened out by the website MEME,corresponding to the conservative motifs P-loop,GLPL,Walker B and RNBS-B of NBS-LRR protein reported in literature.4.UPLC-MS/MS technology was used for qualitative and quantitative detection of resistant secondary metabolites of H.ammodendrum before and after root parasitism.In contrast to the normal growth of H.ammodendron root,the number of differential metabolites in parasitized H.ammodendum roots was 139(93 down-regulated metabolites and 46 up-regulated metabolites).The number of differential metabolites in unparasitized H.ammodendron roots was 146(88 down-regulated metabolites and 58 up-regulated metabolites).47 identical differential metabolites were identified,and most of the metabolites showed the same expression trend.KEGG pathway enrichment of differential metabolites showed that flavonoid biosynthesis and phenylpropane biosynthesis were significantly expressed after parasitism.The resistant metabolites involved in H.ammodendron’s response to C.deserticola parasitic stress can be divided into four types:phytoalexin and allelochemicals may play a role of lethal C.deserticola cells,antioxidant substances combine with reactive oxygen free radicals produced by stress,signal transduction substances transmit parasitic stress signals,lignin and other substances thicken the mechanical barrier of cell wall.To sum up,the resistance of H.ammodendron suffering C.deserticola parasitic stress reaction integrated embodiment of a variety of mechanisms,including the recognition of the invasion signal mechanism,the defense compounds,and the change or form of cell structure,the accumulation of plant hormone regulating function,etc.,among them,the mechanism for identifying the invasion of C.deserticola signal may be a key factor to the result of the parasitism,In this study,two potential resistance genes RGA2-like and RAG3like related to parasitic signal recognition were screened out,which provided a basis for breeding susceptible cultivars of H.ammodendrum. |
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