To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the test...To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry. RESULTSAmong the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC<sub>50</sub> = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G<sub>1</sub> DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphoryla展开更多
Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,w...Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.展开更多
Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically re...Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.展开更多
Plants are constantly exposed to microbial pathogens in the environment.One branch of innate plant immunity is mediated by cell-membrane-localized receptors,but less is known about associations between DNA damage and ...Plants are constantly exposed to microbial pathogens in the environment.One branch of innate plant immunity is mediated by cell-membrane-localized receptors,but less is known about associations between DNA damage and plant immune responses.Here,we show that rice(Oryza sativa)mesophyll cells are prone to DNA double-stranded breaks(DSBs)in response to ZJ173,a strain of Xanthomonas oryzae pv.oryzae(Xoo).The DSB signal transducer ataxia telangiectasia mutated(ATM),but not the ATM and Rad3-related branch,confers resistance against Xoo.Mechanistically,the MRE11–ATM module phosphorylates suppressor of gamma response 1(SOG1),which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection.Intriguingly,overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining(NHEJ)pathway to the alternative NHEJ and homologous recombination pathways atXoo-induced DSBs.The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines.Overall,the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition.The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.展开更多
Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The...Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.展开更多
文摘To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry. RESULTSAmong the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC<sub>50</sub> = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G<sub>1</sub> DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphoryla
基金supported by grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice(Oryza sativa)produces numerous diterpenoid phytoalexins that are important in defense against pathogens.Surprisingly,despite extensive previous investigations,a major group of such phytoalexins,the abietoryzins,were only recently reported.These aromatic abietanes are presumably derived from ent-miltiradiene,but such biosynthetic capacity has not yet been reported in O.sativa.While wild rice has been reported to contain such an enzyme,specifically ent-kaurene synthase-like 10(KSL10),the only characterized ortholog from O.sativa(OsKSL10),specifically from the well-studied cultivar(cv.)Nipponbare,instead has been shown to make ent-sandaracopimaradiene,precursor to the oryzalexins.Notably,in many other cultivars,OsKSL10 is accompanied by a tandem duplicate,termed here OsKSL14.Biochemical characterization of OsKLS14 from cv.Kitaake demonstrates that this produces the expected abietoryzin precursor ent-miltiradiene.Strikingly,phylogenetic analysis of OsKSL10 across the rice pan-genome reveals that from cv.Nipponbare is an outlier,whereas the alleles from most other cultivars group with those from wild rice,suggesting that these also might produce ent-miltiradiene.Indeed,OsKSL10 from cv.Kitaake exhibits such activity as well,consistent with its production of abietoryzins but not oryzalexins.Similarly consistent with these results is the lack of abietoryzin production by cv.Nipponbare.Although their equivalent product outcome might suggest redundancy,OsKSL10 and OsKSL14 were observed to exhibit distinct expression patterns,indicating such differences may underlie retention of these duplicated genes.Regardless,the results reported here clarify abietoryzin biosynthesis and provide insight into the evolution of rice diterpenoid phytoalexins.
基金TI 800/7-1 and TI 800/7-2(SPP 2125 DECRyPT)from the Deutsche Forschungsgemeinschaft,Germany,to A.T.We also acknowledge support from the Cluster of Excellence on Plant Sciences(CEPLAS)the Deutsche Forschungsgemeinschaft under Germany's ExcellenceStrategy-EXC 2048/1-Project ID:390686111and the grant ZU263/11-2(SPP 2125DECRyPT)to A.Z.
文摘Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.
基金supported by the Guangzhou Science and Technology Planning Project (202201010790)the National Natural Science Foundation of China (32188102)+2 种基金the Guangdong Basic and Applied Basic Research Foundation (2023B1515020053)the Youth Innovation of Chinese Academy of Agricultural Sciences (Y20230C36)the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202303).
文摘Plants are constantly exposed to microbial pathogens in the environment.One branch of innate plant immunity is mediated by cell-membrane-localized receptors,but less is known about associations between DNA damage and plant immune responses.Here,we show that rice(Oryza sativa)mesophyll cells are prone to DNA double-stranded breaks(DSBs)in response to ZJ173,a strain of Xanthomonas oryzae pv.oryzae(Xoo).The DSB signal transducer ataxia telangiectasia mutated(ATM),but not the ATM and Rad3-related branch,confers resistance against Xoo.Mechanistically,the MRE11–ATM module phosphorylates suppressor of gamma response 1(SOG1),which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection.Intriguingly,overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining(NHEJ)pathway to the alternative NHEJ and homologous recombination pathways atXoo-induced DSBs.The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines.Overall,the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition.The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.
基金The authors thank Prof.Robert Coates(Univ.Illinois,ret.)for an authentic standard of oryzalexin S.This work was supported by Grants from the NIH(GM131885)and USDA(2020-67013-32557)to R.J.P.
文摘Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.
