| Amorphophallus spp.are important industrial crops in Southwest China.Unfortunately,bacterial diseases are a factor affecting the development of the Amorphophallus spp.industry.Soft rot can be caused by the pathogen Pectobacterium carotovorum subsp.carotovorum(Pcc),leading to large-scale losses in the quality and production of Amorphophallus spp.However,the mechanisms underlying the response of Amorphophallus spp.to Pcc remain largely unknown.This study compared two species of Amorphophallus with different resistance,namely,A.konjac(susceptible)and A.muelleri(resistant),to explore the mechanism of response to Pcc infection by combined transcriptome,metabolome and microbiome analysis.The main results are as follows:1.Gene expression profiling of and A.konjac(susceptible)and A.muelleri(resistant)at different Pcc infection stages were constructed by RNA-Seq.By comparison,23799 and 17125 significant differentially expressed genes(DEGs)in the petiole of A.konjac and A.muelleri were identified after Pcc infection.Individually,3834 DEGs in AK_PC48S(1987 up-regulated and1847 down-regulated),4601 DEGs in AM_PC48S(2179 up-regulated and 2422down-regulated).GO and KEGG enrichment analysis of DEGs revealed that the difference in the response between R and S Amorphophallus spp.lines to Pcc infection at the transcriptional level was mainly reflected in phytohormone signal transduction,plant-pathogen interactions(disease-resistance-related genes,etc.),and metabolism of resistance metabolites.DEGs analysis showed that jasmonic acid(JA)and ethylene(ETH)signal pathways,MAPK signal transduction,pathogenesis-related protein such as PR1 may be major components of the molecular defense resistance mechanisms of the two species of Amorphophallus.41 and 25 hub genes related to defense of A.konjac and A.muelleri were screened by WGCNA analysis,among them,the expression of the MAPK4,MYC2,RTE1,etc.genes were isignificantly upregulated expression after Pcc infection.The results of quantitative real-time PCR(q RT?PCR)of the 15 structural genes were consistent with the RNA-Seq data.At 0 hpi(control),48 hpi and 96 hpi,the expression levels of MPK4 was lower in A.konjac than in A.muelleri.These results suggest that MAPK signal transduction may play an important role in the activation of the immune response in A.muelleri.In addition,JA signaling pathway was involved in the A.konjac and A.muelleri defense response against soft rot disease.Exogenous Me JA treatment has proved that can reduce the soft rot incidence of A.konjac bulbus,and the disease index of Me JA treatment was 13.75%lower than that of control.2.Metabolites in the petiole,root and rhizosphere soil of the two Amorphophallus species were detected by LC?MS/MS nontarget metabolome techniques at various stages of Pcc infection.All of the differentially accumulated metabolites(DAMs)were selected by using multidimensional analysis OPLS-DA and student’s t test.As a result,a total of 391 and 280 DAMs were identified in A.konjac and A.muelleri tissues.KEGG enrichment analysis of DAMs revealed that the DAMs in petiole and root were mainly involved in “phenylpropanoid biosynthesis”,"flavonoid biosynthesis" and "alpha-linolenic acid metabolism" pathway,and DAMs in rhizosphere soil are mainly involved in "plant secondary metabolite biosynthesis pathway".Integration analysis of DEGs and DAMs showed that the key enzyme genes such as PAL,C4 H,4CL and COMT in the phenylpropanoid pathway were significantly upregulated after Pcc infection,meanwhile,some key metabolites(ferulic acid,sinapic acid and syringin)in related metabolic pathways were accumulated.The HCT,C3’H genes may play a key role in regulating the synthesis of caffeoylquinic acid and quercetin.At 48 hpi and 96 hpi,the accumulation of caffeoylquinic acid in A.konjac petiole was down-regulated(0.88 times and0.90 times)compared with the control,whereas it was continuously up-regulated in A.muelleri(1.13 times and 1.30 times).In "alpha-linolenic acid metabolism" pathway,the expression of the LOX2 S gene was significantly upregulated and involved in α-linolenic acid transformation,which promotes the synthesis of methyl jasmonate and its upstream compounds.At 96 hpi,the accumulation of Me JA in the petiole tissue of A.konjac and A.muelleri was 1.36 times and 1.27 times of that of the control,respectively.In vitro bacteriostasis tests of metabolites showed that caffeoylquinic acid,quercetin and kaempferol demonstrated inhibitory effects on the growth of Pcc at concentrations of 25 μg/ml.The results indicate that flavonoids were one of mainly resistance substances of Amorphophallus in response to Pcc stress.3.The microbiome in the petiole,root and rhizosphere soil of the two Amorphophallus species under Pcc stress was evaluated using Illumina Seq.1)In the petiole tissue,co-occurrence network analysis of bacterial communities showed a significant negative correlation between Pectobacterium and Sphingomonas/Microbacterium.The relative abundances of Sphingomonas and Microbacterium in A.konjac were 14.24% and 0.52% at 0 hpi,respectively;these values decreased to 0% at 48 hpi and 96 hpi.The relative abundances of Sphingomonas and Microbacterium in A.muelleri were 63.30% and 4.26% at 0 hpi;these values decreased to 0.11%and 0.01% at 48 hpi and measured 0.75% and 0.05% at 96 hpi,respectively.For the fungal community,under Pcc stress,the relative abundance of Cladosporium was increased in the two species Amorphophallus compared with the control,but the relative abundance in A.konjac was lower than that of A.muelleri.2)Within the root tissue,the relative abundance of Flavobacterium was 3.33%,37.71% and 8.18% in A.konjac at 0 hpi,48 hpi and 96 hpi,respectively,while that of A.muelleri was 1.73%,1.42% and 4.72% at the same time points,respectively.For the fungal community,the relative abundance of Ceratobasidium in A.konjac was decreased continuously after Pcc infection,whereas it was increased continuously in A.muelleri.3)In the rhizosphere soil,the relative abundances of M.alpina,Penicillium,Bacillus and Lysobacter in infected A.konjac plants were lower than those in healthy plants.In contrast,the relative abundances of these microorganisms in infected A.muelleri plants were higher than those in healthy plants.The results of the plate confrontation test showed that the endophytic strains of Cladosporium and M.alpina exerted a strong inhibitory effect on Pcc,with average inhibition bands of 1.08 cm and 1.12 cm,respectively.These results suggest that the dynamic changes and assembly of microbial communities related to Amorphophallus(endophytic microbiome,rhizosphere microbiome)play a role in the response to Pcc stress,with A.muelleri tending to recruit more beneficial microbes to participate in this response.4.The results of metabolome and microbiome correlation analysis showed that phenylpropanoids and flavonoids metabolites in petiole and root tissues,and plant secondary metabolites in soil were positively/negatively correlated with the Amorphophallus-related microbial communities of the two species.In the petiole tissue,Pectobacterium showed a significant negative correlation with the 5-Hydroxyconiferyl alcohol,eriodictyol and naringenin chalcone.In contrast,Cladosporium showed a significant positive correlation with syringin,sinapic acid,ferulic acid and eriodictyol chalcone.Filobasidium showed a significant positive correlation with caffeoylquinic acid and quercetin.In the root tissue,Flavobacterium was significantly positively correlated with the coumarin,eriodictyol and naringenin.In the rhizosphere microbiome,Bacillus was negatively correlated with linolenic acid and positively correlated with octanoate,citric acid and phenylpyruvic acid,whereas Lysobacter exhibited completely opposite correlations;Mortierella was positively correlated with octanoate,pipecolic acid and phenylpyruvic acid.The results showed that plant secondary metabolites were involved in mediating the interaction between microbial communities.In conclusion,under Pcc stress,induction of plant resistance genes,the production of plant secondary metabolites,and the assembly and remodeling of microbial communities,which collectively participate in the Amorphophallus spp.resistance response.This study provides new insights into the “plant-metabolite-microbe” patterns response to pathogen stress. |
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