| Potato scab,one of the main soil-borne diseases of potato,is widespread in the main potato producing areas and the damage level is increasing year by year,which seriously affects the sustainable development of potato industry.In this study,Streptomyces galilaeus 5T-1 was used as the research object.The types of cell wall degrading enzymes produced by S.galilaeus 5T-1,the effects of key pathogenic enzymes on host defense enzymes and the effects of pectin on the metabolism of S.galilaeus 5T-1 were studied.The differential expression of genes during the infection of S.galilaeus 5T-1 was analyzed by transcriptome sequencing.The glycoside hydrolase gene CP966＿RS01990 was cloned and recombinant protein CP966＿RS01990 was purified and verified pathogenic function.The main research results are as follows:1.Cellulase(Cx)and polymethylgalacturonase(PMG)could be produced by S.galilaeus 5T-1 induced by different substrates,and the enzyme activity of PMG(17.79 U/m L)was higher than that of Cx(9.04 U/m L).The diseased potato tissues could produce β-glucosidase(β-D-Glucosidase,β-GLU),Cx and PMG,among whichβ-GLU had high activity(17.61 U/m L),followed by Cx(14.09 U/m L)and PMG(7.72 U/m L).The interaction between S.galilaeus 5T-1 and different potato tissues(tuber flesh and tuber cortex)can cause browning and softening,which can produce Cx,β-GLU and PMG.The enzyme activity of Cx in tuber flesh decreased first,then increased and then decreased,and reached the maximum at 0 h,which was 27.19U/m L.The enzyme activity of Cx in tuber cortex increased first,then decreased and then increased.There were two peaks(7.29 U/m L,9.42 U/m L)at 24 h and 96 h,respectively.The enzyme activity of β-GLU in tuber flesh and tuber cortex showed a rising-decreasing-rising-decreasing trend,and reached the maximum at 48 h and 24 h,respectively,4.59 U/m L and 7.13 U/m L.The enzyme activity of PMG in tuber flesh showed an upward trend,reaching 7.77 U/m L at 144 h.The PMG enzyme activity in tuber cortex showed a dynamic change of first increase,then decrease and then increase,and the maximum value was 12.87 U/m L at 144 h.The above results indicated that Cx and PMG could be produced by both in vivo and in vitro S.galilaeus 5T-1,and there were differences and dynamic changes in the interaction process with different tissues.PMG mainly acted on the late stage of interaction.2、The influencing factors of Cx and PMG enzyme activity from large to small were as follows: initial p H of medium > culture time > temperature > rotation speed.At the same time,the optimal conditions of Cx enzyme production were culture time of 8 d,culture temperature of 23 °C,initial p H of medium of 5,rotation speed of 200 rpm.Under the optimal conditions,the enzyme activity of Cx was 30.47±3.69 U/m L.The optimal conditions for PMG production were incubation time of 6 d,incubation temperature of 23 °C,initial p H of medium of 5,and rotation speed of 200 rpm.Under the optimal conditions for PMG production,the enzyme activity of PMG was42.19 ± 0.56 U/m L.In addition,both Cx and PMG extracted from S.galilaeus 5T-1can promote radish seed germination,and the germination rates were increased by12.69 % and 5 %,respectively.However,the growth of radish seedlings was inhibited,and the plant height was reduced by 50.72 % and 69.52 %,respectively.Moreover,the root and leaf of radish seedlings were diseased,and the incidence rates were92.86 % and 96.43 %,respectively.This indicated that S.galilaeus 5T-1 could produce enzymes that inhibited the growth of plants,and the pathogenicity of PMG was stronger than that of Cx.PMG produced by S.galilaeus 5T-1 could increase the activities of peroxidase,polyphenol oxidase,phenylalanine ammonia lyase,catalase and superoxide dismutase in host plants by 25.48 %–161.05 %,and the difference was significant,indicating that PMG could also stimulate the defense response of host plants.3、A total of 148 differential metabolites were screened from the metabolites of pectin-induced S.galilaeus 5T-1 by hierarchical clustering method,among which the expression levels of 84 metabolites were significantly increased,and the key metabolites were mainly L-Tryptophan,L-Homophenylalanine,2-Aminohexanedioic acid,3-Hydroxyanthranilic acid,Aromatic alcohol,Iminomethylglutamic acid and N-carbamoyl-L-glutamic acid,3-Acylureapropionic acid,and 4-Aminobutyrate ethyl ester.These substances may be involved in the biosynthesis and secretion of pathogenic pectinase by S.galilaeus 5T-1 and the degradation of pectin in cell wall during infection.By KEGG pathway enrichment analysis,there are four pathways that have significant effects on metabolism,β-alanine metabolism,purine metabolism,pantothenate and Co A biosynthesis.4、At 24 h,48 h and 96 h after infection,1319,2853 and 3632 differentially expressed genes were detected in S.galilaeus 5T-1,and the up-regulated genes in each period were respectively enriched in 76,80 and 80 KEGG pathways,including ABC transporter,quorum sensing,carbon metabolism,tricarboxylic acid cycle,glycine,serine and threonine metabolism,amino acid biosynthesis and galactose metabolism.It was found that 341 transcription factor families were involved in the pathogenic process of S.galilaeus 5T-1,and 80 up-regulated genes related to CAZyme were screened.The glycoside hydrolase family and glycosyltransferase family genes accounted for the highest proportion,both 35 %.Finally,20 cellulase and pectinase family genes that may play a key role in the pathogenic process of S.galilaeus 5T-1 infection were identified.5、The glycoside hydrolase gene CP966＿RS01990,which was up-regulated during 5T-1 infection,was cloned.The gene has a conserved domain of GH53 gene family,encoding 519 amino acids with a molecular weight of 54.97 k Da.It belongs to hydrophilic protein.The signal peptide cleavage site is between 31 and 32 amino acids.There is no transmembrane structure,and the subcellular localization is extracellular.The encoded protein has 29.67 % alpha helix,21.97 % extension,7.32 % beta folding,and 41.04 % random coil.The secondary and tertiary structures are composed of secondary and tertiary structures.The prokaryotic expression vector p ET28a-CP966＿RS01990 was constructed,and the recombinant protein was successfully expressed in E.coli BL21(DE3).The recombinant protein was purified by His tag.The recombinant protein CP966＿RS01990 could inhibit the growth and pathogenic function of radish seedlings.The plant height of radish seedlings was reduced by 74.60 % and the incidence was 97.78 %.Based on enzymology,metabolomics and transcriptomics,the production of PMG and Cx from S.galilaeus 5T-1 under in vitro and in vivo has been preliminarily demonstrated,metabolic pathways,key metabolites and key pathogenic enzyme genes were identified in response to pectin induction and host interaction by S.galilaeus5T-1,the glycoside hydrolase gene CP966＿RS01990 was cloned and the pathogenicity of its encoded protein was also verified,revealing the pathogenic mechanism of the cell wall degrading enzymes from S.galilaeus 5T-1.It was confirmed for the first time that cell wall degrading enzymes PMG and Cx were one of the main pathogenic factors of potato scab. |
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