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I-Angustifolius lupine (NLL, Lupinus angustifolius L.) isitshalo semidumba esisetshenziselwa ukukhiqiza ukudla kanye nokwenza ngcono inhlabathi.Ukwandiswa komhlaba wonke kwe-NLL njengesilimo kuhehe isikhunta se-pathogenic esiningi, okuhlanganisa i-lupine anthracnose, ebangela isifo esibhubhisayo se-anthracnose.Ama-alleles amabili, i-Lanr1 kanye ne-AnMan, enikeza ukumelana okukhulayo, asetshenziswe ekuzaleni kwe-NLL, kodwa izindlela eziyisisekelo zamangqamuzana azikaziwa.Kulolu cwaningo, omaka be-Lanr1 kanye ne-AnMan basetshenziswe ukuhlola amasampula e-NLL yaseYurophu.Ukuhlolwa komgomo endaweni elawulwayo kuqinisekise ukusebenza kahle kwabo bobabili abanikeli abangazweli.Ukuchazwa kwephrofayili yenkulumo yofuzo ehlukile kwenziwa emigqeni emele ukumelana neyodwa kanye nethintekayo.Ukumelana ne-anthracnose kwakuhlotshaniswa nokuvezwa ngokweqile kwamagama e-ontology yofuzo “GO:0006952 Defence Response”, “GO:0055114 Redox Process”, kanye “GO:0015979 Photosynthesis”.Ukwengeza, ulayini we-Lanr1(83A:476) ubonise ukuhlelwa kabusha kwe-transcriptome okuphawulekayo ngokushesha ngemva kokujova, kuyilapho eminye imigqa ibonise ukubambezeleka kule mpendulo cishe ngamahora angu-42.Izimpendulo zokuzivikela zihlotshaniswa nezakhi zofuzo ze-TIR-NBS, i-CC-NBS-LRR kanye ne-NBS-LRR, amaprotheni e-10 ahilelekile ku-pathogenesis, amaprotheni okudlulisa i-lipid, i-endoglucan-1,3-β-glucosidase, amaprotheni odonga lwamaseli acebile nge-glycine, kanye nezakhi zofuzo ezivela endleleni esebenzayo ye-oxygen.Izimpendulo zangaphambi kwesikhathi ku-83A:476, okuhlanganisa ukucindezelwa ngokucophelela kwezakhi zofuzo ezihlobene ne-photosynthesis, ziqondane nokuvikelwa okuyimpumelelo phakathi nesigaba sokukhula kwezitshalo sebhayoloji yesikhunta, okuphakamisa ukuthi umphumela udala ukungatheleleki.Ukusabela kwe-Mandeloop kwehliswa ijubane, kanjalo nokuhudula okuvundlile kukonke.
I-lupine enamacembe amancane (NLL, Lupinus angustifolius L.) iwukudla okusamaprotheni amaningi okuvela esifundeni esisentshonalanga yeMedithera1,2.Njengamanje itshalwa njengesitshalo sokudla kwezilwane nabantu.Kuphinde kubhekwe njengomanyolo oluhlaza ezinhlelweni zokuzungezisa izitshalo ngenxa yokulungiswa kwe-nitrogen ngamabhaktheriya alungisa i-symbiotic nitrogen kanye nokuthuthukiswa okuphelele kwesakhiwo senhlabathi.I-NLL iye yabhekana nenqubo esheshayo yokufuywa ekhaya ekhulwini leminyaka elidlule futhi isengaphansi kwengcindezi yokuzalanisa ephezulu3,4,5,6,7,8,9,10,11,12.Ngokutshalwa kabanzi kwe-NLL, ukulandelana kwesikhunta se-pathogenic kwakha izindawo ezintsha zezolimo futhi kwabangela izifo ezintsha ezibhubhisa izitshalo. Okuphawuleka kakhulu kubalimi nabafuyi be-lupine kwaba ukubonakala kwe-anthracnose, ebangelwa ukhunta lwe-pathogenic, i-Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Okuphawuleka kakhulu kubalimi nabafuyi be-lupine kwaba ukubonakala kwe-anthracnose, ebangelwa ukhunta lwe-pathogenic, i-Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного патогенным грибком Colletotrichum (3Boiler) Colletotrichum. Okuphawuleka kakhulu kubalimi belupine nakubafuyi bezilwane kwaba ukuvela kwe-anthracnose ebangelwa ukhunta lwe-pathogenic Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者來说，最引人注目的是炭疽病的起现，它是由病原真菌Colletotrichum Colletotrichum lupini.对于羽扇豆农民和饲养者來说，最引人注目的是炭疽病的出现，它是由病原真菌Colletotrichum lupini). Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, вызываемого патогененым гриtricбком Colleum1 . Okuhlaba umxhwele kakhulu kubalimi nabafuyi be-lupine ukuvela kwe-anthracnose ebangelwa ukhunta oludala i-Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Imibiko yakuqala yalesi sifo yavela eBrazil nase-United States, enezimpawu ezijwayelekile ezibonakala ngo-1912 nango-1929 ngokulandelana.Nokho, ngemva kweminyaka engaba ngu-30, leli gciwane laqanjwa ngokuthi i-Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph Glomerella cingulata (Stoneman) Spauld. & Sacc., телеоморф Glomerella cingulata (Stoneman) Spauld. & Sacc., teleomorph of Glomerella cingulata (Stoneman) Spauld. & Sacc.,有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc.,有目的形态的Glomerella cingulata (Stoneman) Spauld. & Sacc., Glomerella cingulata (Stoneman) Spauld в Целенаправленной морфологии. & Sacc., Glomerella cingulata (Stoneman) Spauld in Targeted Morphology. & H. Schrenk,. & H. Schrenk,.kanye no-H. Schrenk. & H.施伦克，. & H.施伦克，.kanye no-H. Schlenk,.I-phenotyping yesifo sokuqala eyenziwe phakathi nekhulu lama-20 ibonise ukumelana okuthile ku-NLL kanye ne-yellow lupine (L. luteus L.) accessions, kodwa zonke i-lupine emhlophe (L. albus L.) accessions ezihloliwe zazisengozini kakhulu15,16.Ucwaningo luye lwabonisa ukuthi ukuthuthukiswa kwe-anthracnose kuhlotshaniswa nokukhuphuka kwezulu (umswakama womoya) kanye nezinga lokushisa (ebangeni le-12-28 ° C), okuholela ekwephuleni kokumelana emazingeni okushisa aphezulu17, 18. Eqinisweni, isikhathi esidingekayo ukuze i-conidia iqhume futhi isifo siqale , sasifushane izikhathi ezine ku-24 ° C (amahora angu-12) ngaphansi kwezimo eziphakeme ze-12 ° C (amahora angu-12).Ngakho, ukufudumala kwembulunga okuqhubekayo kuye kwaholela ekusakazweni kwe-anthracnose.Kodwa-ke, lesi sifo sabonwa eFrance (1982) nase-Ukraine (1983) njengesandulela sosongo oluzayo, kodwa ngokusobala asizange sinakwa yimboni ye-lupine ngaleso sikhathi20,21.Eminyakeni embalwa kamuva, lesi sifo esibhubhisayo sasakazeka emhlabeni wonke futhi sathinta namazwe amakhulu akhiqiza i-lupine njenge-Australia, Poland neJalimane22,23,24.Kulandela ukuqubuka kwe-anthracnose maphakathi nawo-1990, ukuhlolwa okubanzi kwaholela ekuhlonzweni kwabanikeli abambalwa abangazweli kumasampula e-NLL19.Ukumelana ne-NLL ku-anthracnose kulawulwa ama-alleles amabili ahlukene atholakala emithonjeni ehlukene ye-germplasm: i-Lanr1 ku-cultivar Tanjil kanye ne-Wonga kanye ne-AnMan ku-cultivar.I-Mandalay 25, 26. Lawa ma-allele ahambisana nezimpawu zamangqamuzana ezisekela ukukhethwa kwe-germplasm engazweli ezinhlelweni zokuzalanisa25,26,27,28,29,30.Ulayini wokuzalanisa ongazweli 83A:476 othwele i-Lanr1 allele weqiwe ngomugqa wasendle osengozini u-P27255 ukuze kutholwe inani labantu be-RIL elihlukaniselwa ukumelana ne-anthracnose, okwenze kwaba nokwenzeka ukunikeza i-Lanr1 locus ku-chromosome NLL-1131, 32, 32, 33 yokuxhumanisa i-flankinth markocimic resistance. Uhlaka, i-NLL yembule indawo yawo womathathu ama-alleles ku-chromosome efanayo (NLL-11), kodwa ezikhundleni ezihlukene29,34,35.Kodwa-ke, ngenxa yenani elincane lama-RIL kanye nebanga elikhulu lofuzo phakathi komaka nama-allele ahambisanayo, azikho iziphetho ezithembekile ezingafinyelelwa mayelana nezakhi zawo zofuzo eziyisisekelo.Ngakolunye uhlangothi, ukusetshenziswa kwezakhi zofuzo ezihlehlayo kuma-lupins kunzima ngenxa yamandla awo aphansi kakhulu okuvuselela, okwenza ukuguqulwa kofuzo kube nzima37.
Ukuthuthukiswa kwegciwane elifuywayo elithwala i-allele efiselekayo esimweni se-homozygous, njenge-83A:476 (Lanr1) kanye ne-Mandelup (AnMan), kuvule umnyango wokutadisha ukumelana ne-anthracnose phambi kokuba khona kwezinhlanganisela eziphikisanayo zama-allele kubantu basendle.Amathuba wezinqubo zamangqamuzana.Qhathanisa izimpendulo zokuzivikela ezikhiqizwa ama-genotypes athile.Lolu cwaningo luhlole impendulo yokuqala ye-transcriptome ye-NLL ekugomeni i-C. lupini.Okokuqala, iphaneli yaseYurophu ye-germplasm ye-NLL equkethe imigqa engu-215 yahlolwa kusetshenziswa omaka bemolekyuli abamaka i-Lanr1 ne-AnMan alleles.I-Anthracnose phenotyping yabe yenziwa emigqeni ye-50 NLL, ekhethiwe ngaphambilini kumaka wamangqamuzana, ngaphansi kwezimo ezilawulwayo.Ngokusekelwe kulokhu kuhlolwa, imigqa emine ehlukile ekumelaneni ne-anthracnose kanye nokwakheka kwe-allelic ye-Lanr1/AnMan kukhethelwe ukuchazwa kofuzo lokuvikela okuhlukile kusetshenziswa izindlela ezimbili ezihambisanayo: ukulandelana kwe-RNA ephezulu kanye nokulinganisa kwe-PCR yesikhathi sangempela.
Ukuhlolwa kwesethi ye-NLL germplasm (N = 215) enamaka Lanr1 (Anseq3 kanye ne-Anseq4) kanye ne-AnMan (Anseq4) kanye ne-AnMan (AnManM1) kubonise ukuthi umugqa owodwa kuphela (95726, eduze kwaseSalamanca-b) okhulisa i-alle “yokumelana” kubo bonke omaka, kuyilapho “okungatholakali” kwezimpawu ezitholakala ku-'58 (~73.5%) imigqa.Imigqa eyishumi nantathu ikhiqize ama-alleles amabili "okumelana" kamaka we-Lanr1, futhi imigqa engu-8 ikhiqize ama-alleles "okumelana" we-Lanr1.umaka.I-allele "yokumelana" yomaka we-AnMan (Ithebula Elingeziwe S1).Imigqa emibili bekuyi-heterozygous yomaka we-Anseq3 kanye ne-heterozygous eyodwa yomaka we-AnManM1.Imigqa engu-42 (19.5%) ithwale izigaba eziphambene ze-Anseq3 kanye ne-Anseq4 alleles, okubonisa imvamisa ephezulu yokuhlanganisa kabusha phakathi kwalezi loci ezimbili.I-anthracnose phenotypes ngaphansi kwezimo ezilawulwayo (Ithebula Le-Supplementary S2) yembula ukuhlukahluka kokumelana kwe-genotypes ehloliwe, okuboniswe ngobunzima be-anthracnose.Umehluko kuzikolo ezilinganisiwe wawusukela ku-1.8 (ukumelana ngokumaphakathi) ukuya ku-6.9 (okungelula) kanye nomehluko wesisindo sesitshalo ubusuka ku-0.62 (okululama) ukuya ku-4.45 g (ukumelana). Kube khona ukuhlobana okubalulekile phakathi kwamanani abonwe ekuphindaphindweni okubili kokuhlolwa (i-0.51 yezikolo zobunzima besifo, i-P = 0.00017 kanye ne-0.61 yesisindo sesitshalo, i-P <0.0001) kanye naphakathi kwala mapharamitha amabili (- 0.59 kanye - 0.77, P0100). Kube nokuhlobana okubalulekile phakathi kwamanani aqashelwe kuzimpinda ezimbili zokuhlolwa (i-0.51 yezikolo zokuqina kwesifo, P = 0.00017 kanye no-0.61 ngesisindo sesitshalo, P <0.0001) kanye naphakathi kwala mapharamitha amabili (− 0.59 kanye - 0.700 <0.70 <p. Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперимента (0,51 для баллов тяжезд00, 1 = 6, 1 = массы растения, P < 0,0001), а также между этими двумя параметрами (-0,59 и -0,77, Р < 0,0001) 0,0001). Kutholwe ukuhlobana okubalulekile phakathi kwamanani aqashelwe ekuphindaphindweni okubili kokuhlolwa (0.51 wamaphuzu obunzima besifo, P = 0.00017 kanye no-0.61 wesisindo sesitshalo, P <0.0001), kanye naphakathi kwala mapharamitha amabili (- 0.59 kanye -0.77, P0 <0.0) 0.0).在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分為0.51,P = 111,P = 70.000. 0.0001）以及這两个参数之间（- 0.59 kanye- 0.77,P <0.0001).在 两 次 重复 实验 中 观察 的值 之间 存在 相关性 （疾病 严重 程度 0 评分0 0 p = 10 = 10 = 10 = 10 = 10 = 10 = 10 = 0. , 植物 為 為 0.61 , p <0.0001） 以及 两 个 参数 之间 （（（- 0.59 和– 0.59 和– 0.50 和– 0.50 ,–0 0 7) .– 0 0 7.– 0 0 7 .– 0 0 7 Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тяжести заболевания 0,51, 600, 600 = P <0,0001), futhi между этими двумя параметрами (-0,59 futhi -0,0001) 0,77, P <0,0001. Kube nokuhlobana okubalulekile phakathi kwamanani aqashelwe kuyimpinda (isilinganiso sokuqina kwesifo 0.51, P = 0.00017 nesisindo sesitshalo esingu-0.61, P <0.0001) naphakathi kwalawa mapharamitha amabili (-0.59 kanye -0 .0001) 0.77, P<.0.00 ).Izimpawu ezijwayelekile ezibonwa ezitshalweni ezithintekayo zihlanganisa ukugoba nokusonteka kwesiqu esifana nesakhiwo “somnsalo womalusi”, okulandelwa izilonda ezisaqanda ezine-sporozoite eziwolintshi/ezipinki (I-Supplementary Fig. 1).Ukungena kwe-Australia okuthwele izakhi zofuzo ze-Lanr1 (83A:476 ne-Tanjil) kanye ne-AnMan (Mandelup) zimelana ngokusesilinganisweni, 0.0331 kanye no-0.0036).Eminye imigqa ephinde ibe ne-Lanr1 “engazweli” kanye/noma i-AnMan alleles ikhombisa izimpawu zesifo.
Kuyathakazelisa ukuthi imigqa embalwa ye-NLL entula noma iyiphi i-allele yomaka "engazweli" iveze izinga eliphezulu lokumelana ne-anthracnose (eliqhathanisekayo noma eliphakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani elingu-P <0.0001 kuwo womabili amapharamitha), i-Bojar (inani elingu-P <0.0001 kanye nenani le-Population engu-5 / 9 lesitshalo esingu-700 kanye ne-Population engu-70. 0.0001 ukuthola amaphuzu futhi okungabalulekile ngesisindo). Kuyathakazelisa ukuthi imigqa embalwa ye-NLL entula noma iyiphi i-allele yomaka "engazweli" iveze izinga eliphezulu lokumelana ne-anthracnose (eliqhathanisekayo noma eliphakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani elingu-P <0.0001 kuwo womabili amapharamitha), i-Bojar (inani elingu-P <0.0001 kanye nenani le-Population engu-5 / 9 lesitshalo esingu-700 kanye ne-Population engu-70. 0.0001 ukuthola amaphuzu futhi okungabalulekile ngesisindo). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показали высокий уровень устойчентного е высокий, чем для генотипов Lanr1 или AnMan), таких как Boregine (значение P <0,0001 для обоих параметров), Bojar (значение P < 0,000 0001,0001 стения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Kuyathakazelisa ukuthi imigqa eminingana ye-NLL entula noma iyiphi i-allele kamaka 'engazweli' ibonise izinga eliphezulu lokumelana ne-anthracnose (eqhathaniswa noma ephakeme kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani elingu-P <0.0001 kuwo womabili amapharamitha ), i-Bojar (inani elingu-P <0.0001 ne-7 yesisindo se-7b-0 ye-eva-9 yesitshalo se-0) . Inani elingu-P <0.0001 ukuze lihlolwe futhi alibalulekile ngesisindo).有趣的是，一些缺乏任何“抗性”标记等位基因的NLL 系显示出高水平的炭疽病抗性（帎LanAn1更高），例如Boregine（两个参数的P 值< 0.0001）、Bojar（P 值<得分为0.0001，植物重和量琼卉得（9得（9得（9得（9)值< 0.0001, 重量不显着）. Kuyathakazelisa ukuthi ezinye izinhlelo ze-NLL ezingenazo izimpawu "ze-antigenic" zibonisa ukumelana okuphezulu okuvundlile (okulingana nezakhi zofuzo ze-Lanr1 noma ze-AnMan noma ngaphezulu), njenge-Boregine (zombili ipharamitha P <0.0001), i-Bojar (inani le-P <0.0001, isisindo sesitshalo esingu-0.001) kanye ne-490 yesisindo esibalulekile <0 7b , 9 0 0 0 0 0 0 0 0 000 000 0001) kanye ne-490 yesisindo esibalulekile . Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показали высокие уровни устойчанский чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P для обоих параметров <0,0001), Bojar (значение P <0,0001, масса раст00лц1) и 9 енка P-значение <0,0001, масса незначительна). Kuyathakazelisa ukuthi eminye imigqa ye-NLL engenawo ama-alleles omaka 'wokumelana' abonise amazinga aphezulu okumelana ne-anthracnose (okuqhathaniswa noma ngaphezulu kune-Lanr1 noma i-AnMan genotypes), njenge-Boregine (inani le-P lawo womabili amapharamitha <0.0001) , i-Bojar (inani le-P <0.00000.701, isitshalo se-B9-501, i-P-value <0.00001,701, isitshalo se-B901, i-P-901, i-P-901, i-P-value <0.0001, isisindo asibalulekile).Lesi simo sibonisa ukuthi kungenzeka kube nomthombo wofuzo wenoveli wokumelana, ochaza ukushoda okuphawuliwe kokuhlobana phakathi kwama-marker genotypes kanye ne-phenotypes yezifo (amanani we-P kusuka ku-~0.42 kuya ku-~0.98).Ngakho-ke, ukuhlolwa kwe-Kolmogorov-Smirnov kwabonisa ukuthi idatha yokumelana ne-anthracnose cishe yayisatshalaliswa ngokujwayelekile ukuze kutholakale amaphuzu (ama-P-values ​​0.25 kanye no-0.11) kanye nesisindo sesitshalo (ama-P-values ​​0.47 no-0.55), okusikisela ukuthi ngicabange ukuthi ama-alleles amaningi kune-Lanr1 ne-AnMan ahilelekile.
Ngokusekelwe emiphumeleni yokuhlolwa kokumelana ne-anthracnose, imigqa ye-4 yakhethwa ukuhlaziywa kwe-transcriptome: 83A: 476, i-Boregine, i-Mandelup, ne-Population 22660. Le migqa iphinde yavivinywa ukumelana ne-anthrax ekuhlolweni kwe-inoculation ngokulandelana kwe-RNA, inqobo nje uma ifana nokuhlolwa kwangaphambilini.Amanani wamaphuzu abe kanje: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) kanye nenani labantu abangu-22660 (6.11 ± 1.29 ).
Iphrothokholi ye-Illumina NovaSeq 6000 izuze isilinganiso samapheya e-Mread angu-40.5 ngesampula (29.7 kuya ku-54.4 Mreads) (Ithebula Le-Supplementary S3).Amaphuzu okuqondanisa ochungechungeni lwezithenjwa asukela ku-75.5% ukuya ku-88.6%.Isilinganiso sokuhlobana kwedatha yokubala okufundiwe phakathi kokuhluka kokuhlola phakathi kwezimpinda zezinto eziphilayo kusuka ku-0.812 kuya ku-0.997 (isho u-0.959). Ezakhini zofuzo ezingu-35,170 ezihlaziywe, i-2917 ayizange iveze, kanti ezinye izakhi zofuzo ezingu-4785 zavezwa ezingeni elingenakulinganiswa (isisekelo sisho <5). Ezakhini zofuzo ezingu-35,170 ezihlaziywe, i-2917 ayizange iveze, kanti ezinye izakhi zofuzo ezingu-4785 zavezwa ezingeni elingenakulinganiswa (isisekelo sisho <5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрессировались на ненезнабрессии . Ezakhini zofuzo ezingu-35,170 ezihlaziywe, i-2917 ayizange ibonise inkulumo, futhi izakhi zofuzo ezisele ze-4785 zavezwa ezingeni elingenakulinganiswa (isisekelo sisho <5).在分析的35,170 个基因中，2917 个没有表达，其他4785 个基因的表达可以忽略不计（基本平均值。35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незначительную экспрессировались, а остальные 4785 генов имели незначительную экспрессировались (базение 5). Ezakhini zofuzo ezingu-35,170 ezihlaziywe, i-2917 ayizange iboniswe futhi izakhi zofuzo ezingu-4785 ezisele zinenkulumo enganakwa (isisekelo sisho <5).Ngakho-ke, inani lezakhi zofuzo ezicatshangelwe zivezwe (isisekelo sisho ≥ 5) phakathi nokuhlolwa kwakuyi-27,468 (78.1%) (Ithebula Le-Supplementary S4).
Kusukela ngesikhathi sokuqala, yonke imigqa ye-NLL iphendule ku-inoculation ye-C. lupini (i-strain Col-08) ngokuhlela kabusha i-transcriptome (Ithebula 1), noma kunjalo, umehluko omkhulu ubonwe phakathi kwemigqa.Ngakho-ke, umugqa wokumelana we-83A: 476 (ophethe isakhi se-Lanr1) ubonise ukuhlelwa kabusha kwe-transcriptome okuphawulekayo ngesikhathi sokuqala (i-6 hpi) ngokunyuka kwe-31-69-fold inani lezakhi zofuzo ezihlukanisiwe phezulu naphansi uma kuqhathaniswa namanye amaphuzu esikhathi ngalesi sikhathi.Ngaphezu kwalokho, lesi siqongo sasihlala isikhathi esifushane, njengoba ukubonakaliswa kwezakhi zofuzo ezimbalwa kuphela kwahlala kushintshiwe kakhulu ngesikhathi sesibili (12 hpi).Kuyathakazelisa ukuthi i-Boregine, ephinde yabonisa izinga eliphezulu lokumelana nokuhlolwa kokuxhunyelelwa, ayizange ibhekane nokuhlelwa kabusha kokubhala kabusha okukhulu kangaka ngesikhathi sokuhlolwa.Kodwa-ke, inani lezakhi zofuzo ezivezwe ngokuhlukile (DEG) lalifana ku-Boregine kanye ne-83A: 476 ku-12 HPI.Kokubili i-Mandelup kanye nenani labantu abangu-22660 babonise iziqongo ze-DEG ngesikhathi sokugcina (48 l/s), okubonisa ukubambezeleka okuhlobene kwezimpendulo zokuzivikela.
Ngenxa yokuthi i-83A: I-476 ithole ukuhlelwa kabusha kwe-transcriptome okukhulu ekuphenduleni ku-C. lupini ku-6 HPI uma kuqhathaniswa nayo yonke eminye imigqa, ~ 91% we-DEGs ebonwe ngalesi sikhathi ayeqondile ngokohlu (Fig. 1).Kodwa-ke, kube nokunqwabelana okuthile ezimpendulweni zakuqala phakathi kwemigqa yocwaningo, njengoba u-68.5%, 50.9%, kanye no-52.6% DEG e-Boregine, Mandelup, kanye nenani labantu abangu-22660, ngokulandelana, adlulele nalawo atholakala ku-83A:476 emaphuzwini athile ngesikhathi.Nokho, lawa ma-DEG abala ingxenye encane kuphela (0.97–1.70%) yawo wonke ama-DEG atholwe kusetshenziswa i-83A:476.Ukwengeza, ama-DEG angu-11 avela kuyo yonke imigqa ayehambisana ngalesi sikhathi (Amathebula Engeziwe S4-S6), kuhlanganise nezingxenye ezivamile zezimpendulo zokuzivikela kwezitshalo: iphrotheni yokudlulisa i-lipid (TanjilG_32225), i-endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), i-stress-inducible TaM2_35 iprotheni ye-SAM2G51 kanye ne-TanjilG3538 31), amaprotheni e-latex ayisisekelo (i-TanjilG_32352), kanye namaprotheni amabili odonga lwamaseli esakhiwo anothe nge-glycine (TanjilG_19701 kanye ne-TanjilG_19702).Kuphinde kwaba nokunqwabelana okuphakeme kakhulu ezimpendulweni ze-transcriptome phakathi kuka-83A:476 ne-Boregine ku-24 HPI (inani elingu-16-38% DEG) naphakathi kwe-Mandelup ne-Population 22660 ku-48 HPI (inani elingu-14-20% DEG).
Umdwebo we-Venn obonisa inani lezakhi zofuzo ezivezwe ngokuhlukile (DEG) emigqeni ye-lupine enamaqabunga amancane (NLL) efakwe i-Colletotrichum lupini (uhlobo lwe-Col-08 olutholwe ezinsimini ze-lupine e-Wierzhenice, Poland, 1999).Imigqa ye-NLL ehlaziywe yayiyi: 83A:476 (ekwazi ukumelana, ethwele i-Lanr1 allele), i-Boregine (imelana, isizinda sofuzo asaziwa), i-Mandelup (imelana ngokusesilinganisweni, ithwele i-AnMan allele) kanye nenani labantu abangu-22660 (isengozini kakhulu).Isifinyezo esithi hpi simele amahora ngemva kokugoma.Amanani anguziro akhishiwe ukuze kwenziwe igrafu ibe lula.
Isethi yezakhi zofuzo ezivezwe ngokweqile ku-6 hpi yahlaziywa ukuze kutholakale izizinda zofuzo ze-canonical R (Ithebula Lokwengeza S7).Lolu cwaningo lwembule ukungeniswa kwe-transcriptome kwezakhi zofuzo zokumelana nezifo zakudala ezinezizinda ze-NBS-LRR kuphela ku-83A:476.Leli sethi lalihlanganisa isakhi sofuzo esisodwa se-TIR-NBS-LRR (tanjilg_05042), izinhlobo ezinhlanu zofuzo ze-CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, kanye ne-tanjilg_16162R), Four Tanjilg_16162R, kanye ne-Four NBS-L2 I-RRE (tanjilg_16162) kanye ne-Four NBS-Lrr (tanjilg_16162) kanye ne-Four NBS-LRR (TANJILG_16162).Zonke lezi zakhi zofuzo zinezizinda ze-canonical ezihlelwe ngokulandelana okulondoloziwe.Ngaphezu kwezakhi zofuzo zesizinda se-NBS-LRR, i-RLL kinases eminingana yenziwe yasebenza ku-6 hpi, okuyiyodwa e-Boregine (TanjilG_19877), amabili e-Mandelup (TanjilG_07141 kanye ne-TanjilG_19877) kanye nesibalo sabantu abangu-22660 (TanjilG_068777 kanye ne-TanjilG_09014 kanye ne-2 27: 38 G3)_1 6.
Izakhi zofuzo ezinenkulumo eshintshe kakhulu ekuphenduleni ukujova nge-C. lupini (i-strain Col-08) zafakwa ngaphansi kokuhlaziywa kokunothisa kwe-Gene Ontology (GO) (Ithebula Le-Supplementary S8). Itemu lenqubo yebhayoloji elivame ukumelelwa kakhulu laliyi-"GO:0006952 impendulo yokuvikela" evele kuzinhlanganisela ezingu-6 kwezingu-16 (isikhathi × umugqa) ezinokubaluleka okuphezulu (inani elingu-P <0.001) (Fig. 2). Itemu lenqubo yebhayoloji elivame ukumelelwa kakhulu laliyi-"GO:0006952 impendulo yokuvikela" evele kuzinhlanganisela ezingu-6 kwezingu-16 (isikhathi × umugqa) ezinokubaluleka okuphezulu (inani elingu-P <0.001) (Fig. 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 защитный отвенным термином биологического процесса был «GO: 0006952 защитный отвенным термином биологического процесса был «GO: 0006952 защитный ответ», которлый изя 6 комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Itemu lenqubo yebhayoloji evame ukumelwa kakhulu bekuyi-'GO:0006952 impendulo yokuvikela', evele kuzinhlanganisela ezingu-6 kwezingu-16 (isikhathi × uhlu) ezinokubaluleka okuphezulu (inani elingu-P <0.001) (Fig. 2).最常被过度代表的生物过程术语是“GO:0006952 防御反应”着性 (P 值< 0.001) (图2) Itemu elimele kakhulu lenqubo yebhayoloji lithi “GO:0006952 impendulo yokuvikela”, elivela ku-6 kwezingu-16 (时间×线) izinhlanganisela, ezinokubaluleka okuphezulu (inani elingu-P <0.001) (图2) . Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defence Response», который появлялся 1наий з 6 им с высокой значимостью (значение P <0,001) (рис. 2). Itemu lenqubo yebhayoloji elimelwe kakhulu laliyi-'GO:0006952 Defence Response', elivele kuzinhlanganisela ezingu-6 kwezingu-16 (isikhathi × umugqa) ngokubaluleka okuphezulu (inani elingu-P <0.001) (Fig. 2).Leli gama lalimelwe ngokweqile ngezikhathi ezimbili ku-83A: 476 kanye ne-Boregine (6 kanye ne-24 hpi) futhi ngesikhathi esisodwa e-Mandelup ne-Population 22660 (12 kanye ne-6 hpi, ngokulandelana).Lona umphumela olindelekile, ogqamisa impendulo ye-antifungal yemigqa ephikisayo.Ukwengeza, i-83A: 476 iphendule ku-C. lupini ngokufaka ngokushesha izakhi zofuzo ezihlobene nokuqhuma kwe-oxidative emelelwa igama elithi "GO:0055114 inqubo ye-redox", ebonisa impendulo ethile yokuzivikela, kuyilapho u-Boregine eveza izimpendulo ezithile zokuzivikela, ezihlobene negama elithi 'GO'.:0006950 impendulo yokucindezeleka”.I-Population 22660 ivule impendulo yokumelana evundlile ehilela ama-metabolites esibili, igqamisa inani eleqile lamagama athi “GO:0016104 Inqubo ye-triterpene biosynthesis” kanye ne-“GO:0006722 Inqubo ye-triterpene metabolism” (womabili la magama ayingxenye yeqoqo elifanayo lezakhi zofuzo), kucatshangelwa imiphumela ye-Mandelgil stability kanye ne-GO: 2660. Ngaphezu kwalokho, ukusabela kwangaphambi kwesikhathi 83A:476 (6 hpi) nokubambezeleka kokusabela kwe-Mandelup kanye ne-Population 22660 kufaka phakathi igama elithi GO:0015979 'photosynthesis' nezinye izinqubo zebhayoloji ezihlobene.
Amagama e-bioprocess gene ontology akhethwe esichasiselweni sezakhi zofuzo ezivezwe ngokuhlukile phakathi nezimpendulo ze-transcriptome ze-lupine enamahlamvu amancane (NLL) efakwe i-anthrax lupine (uhlobo lwe-Col-08 olutholwe emasimini e-lupine e-Wierzhenice, e-Poland, ngo-1999) enziwa ihaba kakhulu.Imigqa ye-NLL eyahlaziywa yayiyi: 83A:476 (imelana, ethwele i-homozygous Lanr1 allele), i-Boregine (isizinda sofuzo esingazweli, esingaziwa), i-Mandelup (imelana ngokusesilinganisweni, ethwele i-homozygous AnMan allele) kanye nenani labantu abangu-22660 (abathintekayo).
Ngenxa yokuthi lolu cwaningo luhlose ukuhlonza izakhi zofuzo ezinomthelela ekuphikisweni kwe-anthracnose, izakhi zofuzo ezinikezwe imigomo ethi GO “GO: 0006952 Defensive responses” kanye “GO: 0055114 Redox procedures” zihlaziywe ngokunqanyulwa njengoba isisekelo sisho ≥ 30 okungenani ngomugqa owodwa.× iphuzu ngesikhathi elihlanganisa amanani abalulekile ezibalo we-log2 (ushintsho olusonga).Inani lezakhi zofuzo ezihlangabezana nalezi zindlela zokunquma lalingu-65 ku-GO:0006952 kanye no-524 ku-GO:0055114.
I-83A:476 yembule iziqongo ezimbili ze-DEG ezichasiswe ngegama elithi GO:0006952, eyokuqala kuzakhi zofuzo eziyi-6 iyintshi ngayinye (izakhi zofuzo ezingama-64, umthetho olawulayo okhuphukayo naphansi) kanye nowesibili kumajini angama-24 iyintshi ngayinye (izakhi zofuzo eziyi-15, ukukhuphuka kwemithetho kuphela).I-Boregine iphinde yabonisa ukuthi i-GO:0006952 ifinyelele umvuthwandaba ngesikhathi esifanayo, kodwa nge-DEG encane (11 kanye no-8) nokusebenzisa okuncamelayo.I-Mandeloop ibonise iziqongo ezimbili ze-GO:0006952 ku-12 kanye ne-48 HPI, yomibili iphethe izakhi zofuzo eziyi-12 (eyokuqala enezakhi zofuzo ezisebenzayo, neyesibili enezakhi zofuzo ezicindezelayo kuphela), kuyilapho inani labantu abangu-22660 ku-6 HPI (izakhi zofuzo eziyi-13) libe nokugqama okukhulu kwenani eliphakeme lokwanda.umthethonqubo.Kufanele kuqashelwe ukuthi u-96.4% we-GO:0006952 DEG kulezi ziqongo ube nohlobo olufanayo lokuphendula (phezulu noma phansi), okubonisa ukugqagqana okuphawulekayo kuzimpendulo zokuvikela naphezu kokuhluka kwenani lezakhi zofuzo ezihilelekile.Iqembu elikhulu kunawo wonke lokulandelana okuhlobene negama elithi GO:0006952 lifaka ikhodi ye-Starvation Stress-Associated Message Protein 22 (SAM22-like), engeyesigaba se-10 seprotein ehambisana ne-pathogenesis (PR-10) kanye ne-core protein latex.amaprotheni afanayo (afana ne-MLP) (Fig. 3).La maqembu amabili ahlukene ngesimo sokukhuluma kanye nesiqondiso sempendulo.Izakhi zofuzo ezifaka ikhodi ye-SAM22-like proteins zibonise ukungeniswa okungaguquki nokubalulekile ngesikhathi sokuqala (6 noma i-12 hpi) futhi ngokuvamile ayengaphenduli ekupheleni kokuhlolwa (48 hpi), kuyilapho amaprotheni afana ne-MLP abonisa ukusebenzisana ku-6 hpi.hpi.83A:476 kanye ne-Mandelup ku-48 hp/in, cishe wonke amanye amaphuzu edatha ayengaphenduli.Ukwengeza, umehluko kumaphrofayili wokuvezwa wezakhi zofuzo ezifana ne-SAM22 zamaprotheni ulandele ukuhlukahluka okuphawulwe ekuphikisweni kwe-anthracnose, njengoba imigqa ekwazi ukumelana ne-anthracnose yayinamaphoyinti esikhathi amaningi aheha lezi zakhi zofuzo kakhudlwana kunezakhi zofuzo ezingenwa kalula.Olunye ufuzo lwe-LlR18A/B-like PR-10 lubonise iphethini yenkulumo efana kakhulu nofuzo lweprotheni olufana ne-SAM22.
Izingxenye eziyinhloko zetemu lenqubo yebhayoloji elithi “GO:0006952 Defence Response” kanye namaphethini enkulumo yezakhi zofuzo zekhandidethi ye-Lanr1 ne-AnMan alleles akhonjiwe.Isikali se-Log2 simele amanani we-log2 (ushintsho olugoqiwe) phakathi kokujova (i-Colletotrichum lupini, i-strain Col-08, etholwe emasimini e-lupine, Wizhenica, Poland, 1999) kanye nezitshalo zokulawula (ezifakwe i-sham-inoculated) ngesikhathi esifanayo.Imigqa elandelayo ye-lupine yeqabunga elincane yahlaziywa: 83A:476 (iyamelana, ithwele i-homozygous Lanr1 allele), i-Boregine (imelana, isizinda sofuzo esingaziwa), i-Mandelup (imelana ngokusesilinganisweni, ethwele i-homozygous AnMan allele), kanye ne-Population 22660 (eyathinteka).
Ngaphezu kwalokho, amaphrofayili enkulumo yekhandidethi ye-RNA-seq yofuzo i-Lanr1 (TanjilG_05042) kanye ne-AnMan (TanjilG_12861) ahlolisiswa (Fig. 3).Ufuzo lwe-TanjilG_05042 lubonise ukusabela okubalulekile (ukwenza kusebenze) kokuthi 83A:476 kuphela ngesikhathi sokuqala (6 hpi), kuyilapho i-TanjilG_12861 yayibalulekile ku-Mandeloop kuphela ngezikhathi ezimbili: 6 hpi (umthetho ophansi) kanye no-24 hpi (6 hpi).Nge.).iyalungiseka)).
Izakhi zofuzo ezivezwe kakhulu kuthemu elithi GO: 0055114 "inqubo ye-redox" kwakuyizakhi zofuzo ezifaka ikhodi ye-cytochrome P450 amaprotheni kanye ne-peroxidase (Fig. 4).Kumasampuli ahlukanisiwe ku-83A:476 ku-6 HPI, amanani aphezulu noma aphansi e-log2 (ushintsho olugoqiwe) (ku-86.6% wezakhi zofuzo) ngokuvamile ayebonwa phakathi kwezitshalo ezigonyelwe nezilawulayo, okugqamisa ukusabela okuphezulu kwale genotype ekuzijovweni kocansi.I-83A:476 ibonise i-GO ebaluleke kakhulu: 0055114 DEG ku-6 hpi (503 genes), kuyilapho yonke eminye imigqa iku-48 hpi (Boregine, 31 genes; Mandelup, 85 genes; and Population 22660, 78 genes)).Ezakhini zofuzo eziningi zomndeni we-GO:0055114, izinhlobo ezimbili zezimpendulo zokugoma (ukuqalisa nokuvinjelwa) zabonwa.Kuyathakazelisa ukuthi kufika ku-97.6% we-DEGs akhonjwe igama elithi GO: 0055114 e-Mandelupe ku-48 hp Lokhu kubuka kusikisela ukuthi, naphezu kwesikali esincane kakhulu (okungukuthi, inani lezakhi zofuzo ze-redox eziguquliwe, 85 uma kuqhathaniswa no-503), iphethini yokulibaziseka kokuphendula kwe-transcriptome ku-7 impendulo ye-mandeloup yokuqala ye-mandeloup ye-68.E-Boregine kanye ne-Population 22660, lokhu kuhlangana kuphansi ku-51.6% no-75.6%, ngokulandelana.
Amaphethini okuveza izingxenye eziyinhloko zethemu lenqubo yezinto eziphilayo "GO: 0055114 inqubo ye-Redox" embuliwe.Isikali se-Log2 simele amanani we-log2 (ushintsho olugoqiwe) phakathi kokujova (i-Colletotrichum lupini, i-strain Col-08, etholwe emasimini e-lupine, Wizhenica, Poland, 1999) kanye nezitshalo zokulawula (ezifakwe i-sham-inoculated) ngesikhathi esifanayo.Imigqa elandelayo ye-lupine yeqabunga elincane yahlaziywa: 83A:476 (iyamelana, ithwele i-homozygous Lanr1 allele), i-Boregine (imelana, isizinda sofuzo esingaziwa), i-Mandelup (imelana ngokusesilinganisweni, ethwele i-homozygous AnMan allele), kanye ne-Population 22660 (eyathinteka).
I-83A: 476 Izimpendulo ze-Transcriptomic ku-inoculation nge-C. lupini (i-strain Col-08) iphinde ihlanganise ukuthuliswa okuhlanganisiwe kwezakhi zofuzo ezishiwo kugama elithi GO:0015979 "photosynthesis" nezinye izinqubo ze-biological ezihlobene (FIG. 5).Le sethi ye-GO:0015979 DEG iqukethe izakhi zofuzo ezingu-105 ezicindezelwe kakhulu ku-6 hpi ngo-83A:476.Kulesi sigatshana, izakhi zofuzo ezingama-37 nazo zehliswa e-Mandelup ku-48 HPI kanye nama-35 ngesikhathi esifanayo endaweni yabantu abangu-22660, okuhlanganisa nama-DEG angu-19 avamile kuzo zombili izinhlobo ze-genotype.Awekho ama-DEG ahlobene netemu elithi GO: 0015979 enziwe asebenza ngokuphawulekayo kunoma iyiphi inhlanganisela (umugqa x isikhathi).
Amaphethini okuveza izingxenye eziyinhloko zetemu lenqubo yezinto eziphilayo "GO:0015979 Photosynthesis" embuliwe.Isikali se-Log2 simele amanani we-log2 (ushintsho olugoqiwe) phakathi kokujova (i-Colletotrichum lupini, i-strain Col-08, etholwe emasimini e-lupine, Wizhenica, Poland, 1999) kanye nezitshalo zokulawula (ezifakwe i-sham-inoculated) ngesikhathi esifanayo.Imigqa elandelayo ye-lupine yeqabunga elincane yahlaziywa: 83A:476 (iyamelana, ithwele i-homozygous Lanr1 allele), i-Boregine (imelana, isizinda sofuzo esingaziwa), i-Mandelup (imelana ngokusesilinganisweni, ethwele i-homozygous AnMan allele), kanye ne-Population 22660 (eyathinteka).
Ngokusekelwe emiphumeleni yokuhlaziywa kwenkulumo ehlukile kanye nokubandakanyeka okungenzeka ekuphenduleni kokuzivikela ngokumelene nesikhunta se-pathogenic, le sethi yezakhi zofuzo eziyisikhombisa zakhethwa ukuze kulinganiswe amaphrofayili enkulumo nge-PCR yesikhathi sangempela (Ithebula Le-Supplementary S9).
I- putative protein gene i-TanjilG_10657 iye yathonywa kakhulu kuyo yonke imigqa ehloliwe namaphoyinti esikhathi uma kuqhathaniswa nezitshalo zokulawula (kulingisa) (Amathebula Engeziwe S10, S11).Ngaphezu kwalokho, iphrofayela ye-TanjilG_10657 ibonise ithrendi ekhulayo phakathi nokuhlolwa kwayo yonke imigqa.I-Population 22660 ibonise ukuzwela okuphezulu kakhulu kwe-TanjilG_10657 ku-inoculation nge-activation ephindwe izikhathi ezingu-114 kanye nezinga eliphezulu lokubonisa okuhlobene (4.4 ± 0.4) ku-24 HPI (Fig. 6a).I-PR10 LlR18A protein gene TanjilG_27015 iphinde yabonisa ukusebenza kuyo yonke imigqa namaphoyinti esikhathi, ngokubaluleka kwezibalo kumaphoyinti amaningi wedatha (Fig. 6b).Ngokufanayo ne-TanjilG_10657, izinga eliphezulu lokubonisa okuhlobene le-TanjilG_27015 libonwe ku-22660 labantu abagonyiwe ku-24 HPI (19.5 ± 2.4).I-acid endochitinase gene i-TanjilG_04706 yayimiswe ngokuphawulekayo kuyo yonke imigqa nangazo zonke izikhathi ngaphandle kwe-Boregine 6 hpi (Fig. 6c).Ithonywe ngokuqinile ngesikhathi sokuqala (6 HPI) ku-83A: 476 (ngezikhathi ze-10.5) futhi yanda ngokulinganisela kweminye imigqa (ngezikhathi ze-6.6-7.5).Ngesikhathi sokuhlolwa, inkulumo ye-TanjilG_04706 yahlala kumazinga afanayo ku-83A:476 nase-Boregine, kuyilapho e-Mandelup ne-Population 22660 yanda kakhulu, yafinyelela amanani aphezulu kakhulu (5.9 ± 1.5 kanye no-6.2 ± 1.5, ngokulandelana).I-endoglucan-1,3-β-glucosidase-like gene TanjilG_23384 ibonise ukusebenza okuphezulu ezindaweni ezimbili zokuqala (6 kanye ne-12 hpi) kuyo yonke imigqa ngaphandle kwesibalo sabantu abangu-22660 (Fig. 6d).Amazinga aphezulu wokubonisa ahlobene we-TanjilG_23384 abonwe endaweni yesibili (12 hpi) e-Mandelup (2.7 ± 0.3) kanye no-83A:476 (1.5 ± 0.1).Ku-24 HPI, isisho se-TanjilG_23384 besiphansi ngokuqhathaniswa kuyo yonke imigqa ehloliwe (kusuka ku-0.04 ± 0.009 kuya ku-0.44 ± 0.12).
Amaphrofayili wokuvezwa kwezakhi zofuzo ezikhethiwe (ag) embulwe i-PCR yobuningi.Izinombolo 6, 12 kanye 24 zimele amahora ngemva kokugoma.Izakhi zofuzo ze-LanDExH7 kanye ne-LanTUB6 zisetshenziselwe ukujwayela futhi i-LanTUB6 yasetshenziselwa ukulinganisa kochungechunge oluphakathi.Amabha wamaphutha amele ukuchezuka okujwayelekile okususelwe kuzimpinda ezintathu zebhayoloji, ngayinye eyisilinganiso sokuphindaphinda okuthathu kobuchwepheshe. Ukubaluleka kwezibalo zomehluko kumazinga enkulumo phakathi kokujovwe (i-Colletotrichum lupini, i-strain Col-08, etholwe ngo-1999 kusukela kunkambu ye-lupine e-Wierzenica, e-Poland) kanye nezitshalo zokulawula (i-mock-inoculated) zimakwe ngaphezu kwamaphoyinti edatha (*ivelu engu-P <0.05, **inani elingu-P ≤0 0.0 0). Ukubaluleka kwezibalo zomehluko kumazinga enkulumo phakathi kokujovwe (i-Colletotrichum lupini, i-strain Col-08, etholwe ngo-1999 kusukela kunkambu ye-lupine e-Wierzenica, e-Poland) kanye nezitshalo zokulawula (i-mock-inoculated) zimakwe ngaphezu kwamaphoyinti edatha (*ivelu engu-P <0.05, **inani elingu-P ≤0 0.0 0). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, получен ngo-1999 Плцев, получен в 1999 Плы, полученный в 1999 Пльвый пер. а) и контрольными (ложно инокулированными) растениями отмечена над точками данных (*значение P < 0,05, **значение P ≤ 0,01, *** значение 0, 0. Umehluko obalulekile ngokwezibalo kumazinga okukhuluma phakathi kokujovwe (i-Colletotrichum lupini, i-strain Col-08, etholwe ngo-1999 kusukela endaweni ye-lupine e-Wierzhenice, e-Poland) kanye nezitshalo zokulawula (ezifakwe i-sham-inoculated) ziphawulwe ngaphezu kwamaphoyinti edatha (*inani le-P <0.05, **inani le-P ≤ 0.01 ≤ 0.01 ≤ 0.01 ≤ 0.0 1. ***接种（Colletotrichum lupini，Col-08株，1999年从波兰Wierzenica的羽扇豆田获得异的统计学显着性标记在数据点上方（*P值< 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 （colletorichum lupini , color-08 株 , 1999 年 波兰 波兰 wierzenica 的 羽扇 获得的 统计学 显着性 标记 数据点 上方*p 值 <0.05, **P ≤ 0.01, ***P ≤ 0.001). Статистически значимые различия в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм Col-08, полученный с полей люпи, 9 9 г.) and контрольными (ложно инокулированными) растениями отмечены над точками данных (* значение P < 0,05, ** P-значение ≤ 0,зна70, ***P-01, ***P-0,01, ***P). Umehluko obalulekile ngokwezibalo kumazinga wokukhuluma phakathi kokujovwe (i-Colletotrichum lupini, i-strain Col-08, etholwe ezinsimini ze-lupine e-Verzhenice, e-Poland, ngo-1999) kanye nezitshalo zokulawula (i-sham-inoculated) ziphawulwe ngaphezu kwamaphoyinti edatha (*inani le-P <0.05 , **inani le-P ≤ 0-1 ≤ 0-1 ≤ 0-1 0.Imigqa ye-NLL eyahlaziywa yayiyi: 83A:476 (engazweli, ethwele i-homozygous Lanr1 allele), i-Mandelup (imelana ngokusesilinganisweni, ethwele i-homozygous AnMan allele), i-Boregine (isizinda sofuzo esingazweli, esingaziwa) kanye nenani labantu abangu-22660 (abathintekayo).
Ufuzo lwekhandidethi i-TanjilG_05042 endaweni ye-Lanr1 lubonise iphethini yenkulumo ehluke ngokuphawulekayo kumaphrofayela atholwe ezifundweni ze-RNA-seq (Fig. 6e).Ukusebenza okubalulekile kwalolu fuzo kwabonwa e-Mandelup kanye nenani labantu abangu-22660 (kufika ku-39.7 kanye nezikhathi ezingu-11.7, ngokulandelana), okuholela emazingeni aphezulu kakhulu (kufika ku-1.4 ± 0.14 kanye no-7.2 ± 1.3, ngokulandelana).I-83A:476 iphinde yembula okunye ukulawulwa kofuzo lwe-TanjilG_05042 (kufika ku-3.8-fold), nokho, amazinga enkulumo ehlobene afinyelelwe (0.044 ± 0.002) ayengaphansi ngokuphindwe ka-30 kunalawo abonwa e-Mandelup kanye nenani labantu abangu-22660.kuhlaziywe yi-qPCR kubonise umehluko omkhulu emazingeni enkulumo phakathi kwe-genotypes ezinhlobonhlobo (zokulawula) okugonywe mbumbulu, okufinyelela umehluko ophindwe ka-58 phakathi kwezibalo zabantu 22660 kanye no-83A:476, kanye naphakathi kwezibalo ezingu-22660 nezingu-22660. Umehluko ophindwe kabili wafinyelelwa phakathi kweBoregine ne-Mandalup.
Ufuzo lwekhandidethi endaweni ye-AnMan, i-TanjilG_12861, lwenziwe lwasebenza ukuphendula umgomo ngo-83A:476 kanye ne-Mandelup, lwalungathathi hlangothi kubantu abangu-22660, futhi lwehliselwa ukulawulwa e-Boregine (Fig. 6f).Inkulumo ehlobene yofuzo lwe-TanjilG_12861 yayiphezulu kakhulu ku-83A egonyelwe: 476 (0.14±0.01).I-17.4 kDa class I-heat shock protein gene i-TanjilG_05080 HSP17.4 ibonise amazinga aphansi enkulumo ehlobene kuzo zonke izinhlobo ezifundwayo namaphoyinti esikhathi (Fig. 6g).Inani eliphakeme kakhulu labonwa ku-24 HPI kubantu abangu-22660 (0.14 ± 0.02, ukwanda okuphindwe kasishiyagalombili ekuphenduleni kokugoma).
Ukuqhathaniswa kwamaphrofayela ohlobo lofuzo (Umfanekiso 7) kwembule ukuhlobana okuphezulu phakathi kwe-TanjilG_10657 nezinye izakhi zofuzo ezine: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80G = 0.80) = 0.80 (_9), kanye ne-TanjilG_0.Imiphumela enjalo ingase ibonise ukulawulwa ngokubambisana kwalezi zakhi zofuzo phakathi nezimpendulo zokuzivikela.Izakhi zofuzo ze-TanjilG_12861 kanye ne-TanjilG_23384 zibonise amaphrofayela enkulumo ahlukene anamanani aphansi e-Pearson corelation coefficient (kusuka ku-0.08 kuye ku-0.43 kanye -0.19 kuya ku-0.28, ngokulandelana) uma kuqhathaniswa nezinye izinhlobo zofuzo.
Ukuhlobana phakathi kwamaphrofayili wokuvezwa kofuzo kutholwe kusetshenziswa i-PCR yobuningi.Imigqa elandelayo ye-lupine yeqabunga elincane yahlaziywa: 83A:476 (iyamelana, ithwele i-homozygous Lanr1 allele), i-Mandelup (imelana ngokusesilinganisweni, ithwele i-homozygous AnMan allele), i-Boregine (iyamelana, isizinda sofuzo esingaziwa), kanye ne-Population 22660 (eyathinteka).Amaphuzu esikhathi amathathu abaliwe (amahora angu-6, 12 kanye nangama-24 ngemva kokujova), okuhlanganisa ukujova (i-Colletotrichum lupini, i-strain Col-08, etholwe emasimini e-lupine e-Wierzhenice, e-Poland, ngo-1999) kanye nokulawula (i-sham-inoculated) izitshalo.Isikali sibonisa inani le-Pearson coefficient yokuhlanganisa.
Ngokusekelwe kudatha etholwe kumandla amahhashi angu-6 iyintshi ngayinye, i-WGCNA yenziwe ku-9981 DEG ehlonzwe ngokuqhathanisa izitshalo ezigonyelwe nezilawulayo ukuze kugxilwe ezimpendulweni zokuvikela zangaphambi kwesikhathi (Ithebula Le-Supplementary S12).Amamojula ofuzo angamashumi amabili nambili (amaqoqo) atholwe anamaphrofayela enkulumo ehlobene (enhle noma embi) phakathi kwe-genotype nokuhluka kokuhlola. Ngokwesilinganiso, amazinga enkulumo yofuzo ayehla ngokulandelana 83A:476 > Mandelup > Boregine > Population 22660 (kokubili okuhlukile, nokho, le nqubo ibinamandla ezitshalweni zokulawula). Ngokwesilinganiso, amazinga enkulumo yofuzo ayehla ngokulandelana 83A:476 > Mandelup > Boregine > Population 22660 (kokubili okuhlukile, nokho, le nqubo ibinamandla ezitshalweni zokulawula). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 indawo). Ngokwesilinganiso, amazinga wokuvezwa kwezakhi zofuzo ehle ngohlelo 83A:476 > Mandelup > Boregine > Population 22660 (kokubili okuhlukile, nokho, lo mkhuba ubunamandla ezitshalweni zokulawula).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660 的顺序下降（然而，在两种种种种中变公物更强.平均 而 言 , 基因 水平 按 83a: 476> mandelup> boregine> inani labantu 22660 的顺序 下降 （，在 种 中 （，在 种 中 （，在 种 中 ） ........ ........ ........ В среднем уровни экспрессии генов снижались в ряду 83A:476 Mandelup > Boregine > Population 22660 futhi). Ngokwesilinganiso, amazinga wokuvezwa kwezakhi zofuzo ehle ochungechungeni 83A:476 > Mandelup > Boregine > Population 22660 (nokho, kuzo zombili izinhlobo, lo mkhuba ubunamandla ezitshalweni zokulawula).Ukugonywa kubangele ukulawulwa kokubonakaliswa kofuzo, ikakhulukazi kumamojula 18, 19, 14, 6 kanye no-1 (ngokuhlelwa komphumela), ukulawulwa okungalungile (isb amamojula 9 no-20) noma ngemiphumela engathathi hlangothi (isb amamojula 11, 22, 8 kanye ne-13).Ukuhlaziywa kwethemu lokucebisa le-GO (Ithebula Lokwengeza S13) kwembule okuthi “GO: 0006952 Izimpendulo ezivikelayo” zemojula egonyelwe (18) esebenzisa ubuningi, okuhlanganisa ufuzo oluhlaziywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10105G izithombe eziningi njenge-sunjilG_1065G) amamojula e-synthesis (9).I-concentrator ye-Module 18 (Fig. 8) ihlonzwe njengofuzo lwe-TanjilG_26536 olufaka ikhodi ye-PR-10-like LlR18B protein, futhi i-module 9 concentrator ikhonjwe njengofuzo lwe-TanjilG_28955 olufaka ikhodi ye-photosystem II PsbQ protein.I-anthracnose resistance gene i-Lanr1, i-TanjilG_05042, itholwe kumojula 22 (Fig. 9) futhi ihlotshaniswa namagama athi "GO:0044260 Cellular macromolecular metabolic procedures" kanye "GO:0006355 Transcriptional regulation, DNA templating1G_0 ephethe i-Tag.isakhi sofuzo sifaka i-heat stress transcription factor A-4a (HSFA4a).
Ukuhlaziywa kwenethiwekhi okunesisindo sokuchazwa kofuzo ngokuhlanganyela kwamamojula anamagama enqubo yebhayoloji amelwe kakhulu “GO: 0006952 Defence responses”.I-Ligation yenziwe lula ukuze kugqanyiswe izakhi zofuzo ezine ezihlaziywe yi-qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 kanye ne-TanjilG_27015).
Ukuhlaziywa kwenethiwekhi okunesisindo somusho wofuzo wemojuli enegama lenqubo yebhayoloji elimelele kakhulu elithi “GO: 0006355: Umthetho obhaliwe, ukulinganisa kwe-DNA” kanye nokuthwala uhlobo lokumelana ne-anthracnose i-Lanr1 TanjilG_05042.I-Ligation yenziwe lula ukuze kuhlukaniswe ufuzo lwe-TanjilG_05042 kanye nofuzo olumaphakathi lwe-TanjilG_01212.
Ukuhlolwa kokumelana ne-anthracnose okuqoqwe e-Australia kwabonisa ukuthi iningi lama-cultivar asheshe akhululwa ayesengozini;I-Kalya, i-Coromup ne-Mandelup zichazwe njengezimelana ngokusesilinganisweni, kuyilapho i-Wonga, i-Tanjil ne-83A:476 zichazwe njengezingazweli kakhulu26,27,31.yayine-allele efanayo yokumelana, eqokwe i-Lanr1, futhi i-Coromup ne-Mandelup yaba ne-allele ehlukile, eqokwe i-AnMan10, 26, 39, kuyilapho u-Kalya edlulisela i-allele ehlukile., Lana2.Ukuhlolelwa ukumelana ne-anthracnose eJalimane kuholele ekuhlonzweni komugqa ongazweli we-Bo7212 onekhandidethi i-allele ngaphandle kwe-Lanr1, eqokiwe i-LanrBo36.
Ucwaningo lwethu lwembula imvamisa ephansi kakhulu (cishe i-6%) ye-Lanr1 allele ku-germplasm ehloliwe.Lokhu kubuka kuhambisana nemiphumela yokuhlola i-germplasm yaseMpumalanga Yurophu kusetshenziswa izimpawu ze-Anseq3 ne-Anseq4, ezibonise ukuthi i-Lanr1 allele ikhona emigqeni emibili kuphela yaseBelarusian.Lokhu kusikisela ukuthi i-Lanr1 allele ayikasetshenziswa kabanzi izinhlelo zendawo zokuzalanisa, ngokungafani ne-Australia, lapho ingenye yezindlela eziyinhloko zokuzalanisa okusizwa umaka.Lokhu kungase kube ngenxa yezinga eliphansi lokumelana elihlinzekwe yi-Lanr1 allele ezimeni zenkambu yaseYurophu uma kuqhathaniswa nombiko wase-Australia.Ukwengeza, izifundo ze-anthracnose ezindaweni ezinemvula ephezulu e-Australia zibonise ukuthi izimpendulo zokumelana ne-Lanr1 allele zingase zingasebenzi ezimweni zezulu ezivumela ukukhula nokuthuthukiswa okusheshayo kwe-pathogen19,42.Eqinisweni, ocwaningweni lwamanje, ezinye izimpawu ze-anthracnose nazo zabonwa kuma-genotypes aphethe i-Lanr1 allele, okuphakamisa ukuthi ukumelana kungase kunyamalale ngaphansi kwezimo ezikahle zokuthuthukiswa kwe-C. lupini.Ngaphezu kwalokho, ukuhumusha okungamanga okuhle kokuba khona komaka be-Anseq3 kanye ne-Anseq4, okungaba ngu-1 cm ukusuka endaweni ye-Lanr1, kungenzeka kube ngu-28,30,43.
Ucwaningo lwethu lubonise ukuthi i-83A: i-476, ephethe i-Lanr1 allele, iphendule ku-C. lupini inoculation nge-reprogramming enkulu ye-transcriptome ngesikhathi sokuqala esihlaziywe (6 hpi), kuyilapho e-Mandelup, ephethe i-AnMan allele, izimpendulo ze-transcriptomic zabonwa kamuva kakhulu.(kusuka ku-24 kuye ku-48 hp).Lokhu kuhlukahluka kwesikhashana ekuphenduleni kokuzivikela kuhlotshaniswa nokuhluka kwezimpawu zesifo, okugqamisa ukubaluleka kokuqashelwa kwe-pathogen kusenesikhathi ukuze kuphendule ngempumelelo ukumelana.Ukuthelela izicubu zezitshalo, izinhlamvu ze-anthrax kufanele zidlule ezigabeni ezimbalwa zokukhula endaweni yesitshalo, okuhlanganisa ukuhluma, ukuhlukana kwamaseli, nokwakheka kwe-appressorium.I-appendage isakhiwo esithathelwanayo esinamathela ebusweni bomsingathi futhi senze kube lula ukungena kumathishu abambayo.Ngakho-ke, izinhlamvu ze-C. gloeosporioides ekukhishweni kwe-pea zibonise ukuhlukaniswa kokuqala kwe-nucleus ngemva kwemizuzu engu-75-90 yokufakelwa, ukwakheka kwe-tube yegciwane ngemva kwemizuzu engu-90-120, nokucindezela ngemva kwamahora angu-4 45.I-Mango C. gloeosporioides ibonise ukuhluma kwe-condial engaphezu kuka-40% ngemva kwamahora angu-3 ekufukamela kanye nokwakheka okungama-20% kwama-appressors ngemva kwamahora angu-4.I-CAP20 ehlobene ne-virulence yofuzo lwe-C. gloeosporioides ibonise umsebenzi wokuloba ku-epiphyte-forming conidia ngemva kwamahora angu-3.5 ekufukamele ku-avocado surface wax nokugxila okuphezulu kwe-CAP20 amaprotheni ngemva kwamahora angu-4 46 min.Ngokufanayo, umsebenzi wezakhi zofuzo ze-melanin biosynthesis ku-C. trifolii wenziwa phakathi ne-incubation yamahora angu-2 elandelwa ukwakhiwa kwe-appressorium ngemva kwehora elingu-1.Ucwaningo lwezicubu zamaqabunga lubonise ukuthi ama-strawberries afakwe nge-C. acutatum anokucindezela kokuqala ku-8 hpi, kanti utamatisi ofakwe nge-C. coccodes unokucindezela kokuqala ku-4 hpi48,49.ngokuyinhloko ihambisana nesilinganiso sesikhathi se-Colletotrichum spp.inqubo ezithathelwanayo.Izimpendulo ezisheshayo zokuzivikela ku-83A: 476 ziphakamisa ukubandakanyeka kokumelana nezitshalo kanye nezakhi zofuzo zokuzivikela ezibangelwa umphumela (ETI) kulo mugqa, kuyilapho izimpendulo ezibambezelekile ze-Mandelup zisekela i-micro-associated molecular pattern-triggered immune (MTI) hypothesis 50. Izimpendulo zangaphambili ku-83A: 476 kanye ne-Mandelup.Ukunqwabelana okuyingxenye phakathi kwezakhi zofuzo ezilawulwa phezulu noma phansi ekuphenduleni okubambezelekile nakho kusekela lo mqondo, njengoba i-ETI ivame ukubhekwa njengempendulo ye-MTI esheshayo futhi ethuthukisiwe efinyelela umvuthwandaba wokufa kwamangqamuzana ahleliwe endaweni yokutheleleka, eyaziwa ngokuthi ukushaqeka kwe-anaphylactic 51,52.
Izakhi zofuzo eziningi ezishiwo ngegama elimele kakhulu i-Gene Ontology GO:0006952 “Impendulo Yokuvikela” ama-homologue angu-11 omlayezo wokuzila ukudla obangelwa ingcindezi engu-22 protein (efana ne-SAM22) kanye nokuthandwa kakhulu kwe-latex protein-likes (MLPs).-njengamaprotheni 31, 34, 43 no-423 abonise ukulandelana kokufana.Izakhi zofuzo ezifana ne-SAM22 zibonise ukusebenza okuphawulekayo okuthathe isikhathi eside, okubonisa amazinga akhulayo okumelana ne-anthracnose (83A: 476 kanye ne-Boregine).Nokho, izakhi zofuzo ezifana ne-MLP zehliswa kuphela emigqeni ephethe i-allele yokumelana nekhandidethi (83A:476/Lanr1 ku-6 hpi kanye ne-Mandelup/AnMan ku-24 hpi).Kufanele kuqashelwe ukuthi wonke ama-homologue afana ne-SAM22 ahlonziwe asuka kuqoqo lofuzo oluhlanganisa cishe u-105 kb, kuyilapho ufuzo olufana ne-MLP luvela ezifundeni ezihlukene ze-genome.Ukusebenza okudidiyelwe kwezakhi zofuzo ezifana ne-SAM22 kuphinde kwatholakala esifundweni sethu sangaphambilini sokumelana ne-NLL ku-Diaporthetoxica inoculation, okuphakamisa ukuthi bahileleke ezingxenyeni ezivundlile zempendulo yokuzivikela.Lesi siphetho siphinde sisekelwe imibiko yokusabela okuhle kwezakhi zofuzo ezifana ne-SAM22 ekulimaleni noma ekwelapheni nge-salicylic acid, ama-inducers esikhunta, noma i-hydrogen peroxide.
Izakhi zofuzo ezifana ne-MLP zikhonjisiwe ukuthi zisabela ekucindezelekeni okuhlukahlukene kwe-abiotic kanye ne-biotic, okuhlanganisa amagciwane, amagciwane kanye nezifo zokukhunta ezibangelwa amagciwane ezinhlotsheni eziningi zezitshalo55.Izikhombisi-ndlela zokusabela ekusebenzelaneni okuthile phakathi kwezitshalo namagciwane zazisukela ekwandeni kakhulu (okungukuthi, ngesikhathi sokugcwele ukotini ne-Verticillium dahliae) kuya ekwehleni kakhulu (okungukuthi, ngemva kokutheleleka kwe-apula nge-Alternaria spp.)56,57.Ukwehliswa okuphawulekayo kofuzo lwe-MLP-like 423 kuye kwabonwa ngesikhathi sokuvikela ukwatapheya ekuthelelekeni kwe-F. niger nangesikhathi sokutheleleka kwesihlahla sama-apula i-Botryosphaeria berengeriana f.cn.I-piricola kanye ne-Alternaria alternata yi-apula pathotypes58,59.Ukwengeza, i-apple calli egcizelela kakhulu isakhi sofuzo esifana ne-MLP-like 423 yayinokubonakaliswa okuphansi kwezakhi zofuzo ezihambisana nokuphikiswa futhi yayisengozini yokungenwa yisikhunta59.Kulandela i-Fusarium oxysporum f, i-MLP-like 423 gene nayo yacindezelwa ku-germplasm kabhontshisi ovamile ongazweli.cn.Ukutheleleka kukabhontshisi 60.
Amanye amalungu omndeni we-PR-10 ahlonzwe ocwaningweni lwethu lwe-RNA-seq kwakuyizakhi zofuzo ze-LlR18A ne-LlR18B ekuphenduleni ukulawulwa, kanye nofuzo olumisiwe (1) noma olwehlisiwe (izakhi ezi-3) zeprotheni yokudlulisa i-lipid i-DIR1..Ngaphezu kwalokho, i-WGCNA igqamisa isakhi sofuzo se-LlR18B njengehabhu kule mojula, esengozini enkulu yokugonywa futhi ephethe izakhi zofuzo ezimbalwa zokuzivikela.Izakhi zofuzo ze-LlR18A kanye ne-LlR18B zafakwa emaqabunga e-lupine aphuzi ngokuphendula amagciwane e-pathogenic, kanye neziqu ze-NLL ngemva kokufakwa kwe-D. toxica inoculation, kuyilapho i-rice homologue yalezi zakhi zofuzo, i-RSOsPR10, yabangelwa ukutheleleka kwefungal okungenzeka ukuthi kuhileleke ku-jasmonic acid, 62, 62, i-DIncodesic, i-DINCO, i-DINC. amaprotheni okuthutha ama-lipid adingekayo ukuze kuqale ukuphikiswa okutholakala kwesistimu (SAR).Ngokuthuthuka kokusabela kokuvikela, iphrotheni ye-DIR1 ithuthwa isuka lapho kugxilwe khona ukutheleleka nge-phloem ukuze ifake i-SAR ezithweni ezikude.Kuyathakazelisa ukuthi ufuzo lwe-TanjilG_02313 DIR1 lwaqanjwa ngokuphawulekayo endaweni yokuqala emigqeni 84A:476 kanye Nenani labantu 22660, kodwa ukumelana ne-anthracnose kuthuthuke ngempumelelo kuphela kumugqa 84A:476.Lokhu kungase kubonise ukungasebenzi okuthile kofuzo lwe-DIR1 ku-NLL, njengoba ama-homologue amathathu asele aphendule ekugomeni kuphela kumugqa we-83A:476 ku-6 hpi, futhi le mpendulo iqondiswe phansi.
Ocwaningweni lwethu, izakhi ezivame kakhulu ezihambisana nenqubo yezinto eziphilayo ebizwa ngokuthi “GO:0055114 Redox process” kwakuyi-cytochrome P450 protein, peroxidase, linoleic acid 9S-/13S-lipoxygenase, kanye ne-1-aminocyclopropane-1-carboxylic acid oxidase.Ngaphezu kwalokho, i-WGCNA yethu ichaza i-homologue ye-HSFA4a njengehabhu ephethe amamojula afana nekhandidethi lofuzo lokumelana ne-Lanr1 i-TanjilG_05042.I-HSFA4a iyingxenye yomthetho oncike ku-redox wokulotshwa kwe-nuclear ezitshalweni.
Amaprotheni e-Cytochrome P450 angama-oxidoreductases akha i-NADPH kanye/noma i-hydroxylation encike ku-O2 ku-metabolism eyinhloko neyesibili, okuhlanganisa i-metabolism ye-xenobiotics, kanye namahomoni, ama-fatty acids, ama-sterols, izakhi zodonga lwamaseli, ama-biopolymers, kanye ne-biosynthesis ye-protective compounds ku-P50 encishisiwe isitshalo se-P5, i-variosynthesis ye-protective ye-P5, i-P400 yocwaningo lwe-P5. 10.6 log2 (ushintsho oluphindwe kabili) ukuya ku-5.7 ngenxa yenani elikhulu lama-homologue ashintshiwe (37) kanye nomehluko wamaphethini okuphendula phakathi kofuzo oluthile, okubonisa ukubukezwa okungaphezulu..Ukusebenzisa idatha ye-RNA-seq kuphela ukuze kucaciswe umsebenzi webhayoloji obekayo wezakhi zofuzo ze-NLL kumndeni omkhulu wamaprotheni kungaba ukuqagela kakhulu.Kodwa-ke, kufanele kuqashelwe ukuthi ezinye izakhi zofuzo ze-cytochrome P450 zihlotshaniswa nokwanda kokumelana nesikhunta se-pathogenic noma amagciwane, kuhlanganise negalelo ekuphenduleni kwe-allergies69,70,71.
Ama-peroxidase e-Class III ama-enzyme ezitshalo asebenzayo ahilelekile ezinhlobonhlobo zezinqubo ze-metabolic ngesikhathi sokukhula nokukhula kwezitshalo, kanye nokuphendula izingcindezi zemvelo ezifana nosawoti, isomiso, ukukhanya okuphezulu, nokuhlasela kwe-pathogen72.I-Peroxidase ibandakanyeka ekusebenzelaneni kwezinhlobo zezitshalo eziningana ne-Anthracis, okuhlanganisa i-Stylosanthes humilis ne-C. gloeosporioides, i-Lens culinaris ne-C. truncatum, i-Phaseolus vulgaris ne-C. lindemuthianum, i-Cucumis sativus ne-C. lagenarium73,74,75.75.Impendulo ishesha kakhulu, ngezinye izikhathi ngisho naku-4 HPI, ngaphambi kokuba ukhunta lungene esicutshini sesitshalo73.Ufuzo lwe-peroxidase luphinde lwasabela ekufakweni kwe-D. toxica NLL.Ngaphezu kwemisebenzi yabo evamile yokulawula ukuqhuma kwe-oxidative noma ukuqeda ukucindezeleka okwenziwe nge-oxidative, ama-peroxidase angaphazamisa ukukhula kwe-pathogen ngokudala izithiyo zomzimba ezisekelwe ekuqinisweni kodonga lwamaseli ngesikhathi sokuhlanganiswa, i-subunit noma ukuxhumanisa okuphambene kwezinhlanganisela ezithile.Lo msebenzi ungabalulwa ku-silico kuhlobo lofuzo lwe-TanjilG_03329 olufaka i-puative lignin-forming anion peroxidase eyaye yaqondiswa kakhulu ocwaningweni lwethu ku-83A:476 umugqa wokumelana no-6 HPI, kodwa hhayi kwezinye izinhlobo namaphoyinti esikhathi angazange aphendule.
I-9S-/13S-lipoxygenase ye-linoleic acid iyisinyathelo sokuqala sendlela ye-oxidative ye-lipid biosynthesis78.Imikhiqizo yale ndlela inemisebenzi eminingi ekuvikelweni kwezitshalo, okuhlanganisa ukuqiniswa kwezindonga zamangqamuzana ngokwakhiwa kwe-callose ne-pectin deposits, nokulawulwa kokucindezeleka okwenziwe nge-oxidative ngokukhiqizwa kwezinhlobo ze-oxygen esebenzayo79,80,81,82,83.Ocwaningweni lwamanje, ukuvezwa kwe-linoleic acid 9S-/13S-lipoxygenase kwashintshwa kuzo zonke izinhlobo, kodwa kubantu abathintekayo abangu-22660, ukulawulwa kwandile ngezikhathi ezihlukene, kuyilapho ezinkingeni ezithwala i-Lanr1 engazweli kanye ne-AnMan allele, igcizelela ukuhlukahluka kwe-oxylipinnth reaction phakathi kwalezi zingqimba ze-oxylipinnotype.
I-homologue ye-1-aminocyclopropane-1-carboxylate oxidase (ACO) iphakanyiswe kakhulu (izakhi zofuzo eziyisi-9) noma yehliswa (izakhi zofuzo ezi-2) lapho ifakwe i-lupine.Ngaphandle kokubili, zonke lezi zimpendulo zenzeke ku-6 hp.ngo83:476.Ukusabela kwe-enzymatic okuphakathi kwamaprotheni e-ACO kuyisinyathelo sokunciphisa izinga ekukhiqizeni i-ethylene ngakho-ke kulawulwa kakhulu84.I-Ethylene iyi-hormone yesitshalo edlala izindima ezihlukahlukene ekulawuleni ukukhula kwezitshalo kanye nokusabela ezimweni ze-abiotic kanye ne-biotic stress.Ukufakwa kokubhalwa kwe-ACO kanye nokusebenza kwendlela yokubonisa i-ethylene kuhileleke ekwandiseni ukumelana nelayisi ku-hemibiotrophic fungus oryzae oryzae ngokulawula ukukhiqizwa kwezinhlobo ze-oksijini ezisebenzayo kanye nama-phytoalexins.Inqubo yokutheleleka kweqabunga efana kakhulu etholakala phakathi kwe-M. oryzae kanye ne-C. lupini88,89, ngokumelene nesizinda sokukhuphuka okuphawulekayo kwama-homologues e-ACO ku-83A: umugqa we-476 obikwe kulolu cwaningo, ishintsha amathuba okunikeza ukumelana ne-NLL anthracnose I-Ethylene isinyathelo esimaphakathi esibonisa izimpawu ezindleleni zamangqamuzana.
Ocwaningweni lwamanje, ukucindezelwa okukhulu kwezakhi zofuzo eziningi ezihlobene ne-photosynthesis kubonwe ku-6 hpi ngo-83A:476 kanye naku-48 hpi eMandeloop kanye nenani labantu abangu-22660.Izinga nokuqhubeka kwalezi zinguquko kuhambisana nezinga.Ukumelana ne-anthracnose kwabonwa kulolu cwaningo.Muva nje, ukucindezelwa okuqinile nokungaphambi kwesikhathi kwemibhalo ehlobene ne-photosynthesis kuye kwabikwa kumamodeli amaningana okusebenzelana kwezitshalo-pathogen, okuhlanganisa amagciwane e-pathogenic kanye nesikhunta.Ukushesha (kusuka ku-2 HPI kokunye ukusebenzisana) kanye nokucindezelwa komhlaba wonke kwezakhi zofuzo ezihambisana ne-photosynthesis ekuphenduleni ukutheleleka kungabangela ukuzivikela kwezitshalo ngokusekelwe ekusetshenzisweni kwezinhlobo ze-oksijini ezisebenzayo kanye nokusebenzisana kwazo nendlela ye-salicylic acid ukuze kulamule ukungezwani komzimba 90,94.
Sengiphetha, izindlela zokuphendula zokuvikela ezihlongozwayo zozalo olumelana kakhulu (83A:476) zifaka ukubonwa kwe-pathogen okusheshayo yi-R gene (okungenzeka ukuthi i-TIR-NBS-LRR TanjilG_05042) kanye ne-allergic reaction-mediated salicylic acid kanye nokusayina kwe-ethylene, okulandelwa ukusungulwa kwe-SAR yebanga elide.isenzo sisekelwa iphrotheni ye-DIR-1.Kufanele kuqashelwe ukuthi isikhathi se-biotrophic sokutheleleka kwe-C. lupini sifushane kakhulu (cishe izinsuku ezi-2), kulandelwa ukukhula kwe-necrotic95.Ukuguquka phakathi kwalezi zigaba kungase kuhlotshaniswe ne-necrosis kanye nokuvezwa kwamaprotheni e-ethylene-inducible asebenza njengezinto ezibangela ukusabela kwe-hypersensitivity ezitshalweni eziphethe.Ngakho-ke, iwindi lesikhathi lokuthunjwa ngempumelelo kwe-C. lupini esigabeni se-biotrophic lincane kakhulu.Ukuhlelwa kabusha kwezakhi zofuzo ezihlotshaniswa ne-redox ne-photosynthesis okuphawulwe ku-83A:476 ku-6 hpi kuhambisana nokuqhubeka kwe-fungal hyphae futhi kwazisa ukuthuthukiswa kokusabela okuphumelelayo kokuvikela esigabeni se-biotrophic.Izimpendulo ze-transcriptomic ze-Mandelup kanye nenani labantu abangu-22660 zingase zibambezeleke kakhulu ukubamba ukhunta ngaphambi kokushintshela ekukhuleni kwe-necrotic, nokho, i-Mandelup ingase isebenze kakhulu kunabantu abangu-22660 ngoba ukulawulwa okushesha kakhulu kwephrotheni ye-PR-10 kukhuthaza ukumelana okuvundlile.
I-ETI, eshayelwa isakhi sofuzo esingu-R, ibonakala iyindlela evamile yokumelana nobhontshisi ku-anthracnose.Ngakho, kumodeli ye-legume ye-Medicago truncatula, ukumelana ne-anthracnose kunikezwa isakhi sofuzo se-RCT1, ilungu lesigaba sofuzo se-TIR-NBS-LRR97 yesitshalo se-R.Lesi sakhi sofuzo siphinde sinikeze ukumelana kwe-anthracnose okubanzi kwe-alfalfa lapho sidluliselwa ezitshalweni ezithintekayo.Kubhontshisi ojwayelekile (P. vulgaris), ngaphezu kweshumi nambili izakhi zofuzo zokumelana ne-anthracnose zihlonziwe kuze kube manje.Ezinye zalezi zakhi zofuzo zitholakala ezifundeni ezingenazo izinhlobo zofuzo ze-canonical R, nokho ezinye eziningi zitholakala emaphethelweni ama-chromosome aphethe iqoqo lofuzo le-NBS-LRR, okuhlanganisa i-TIR-NBS-LRRs99.Ucwaningo lwe-SSR olubanzi lwe-genome luphinde lwaqinisekisa ukuhlangana kofuzo lwe-NBS-LRR nokumelana ne-anthracnose kubhontshisi ovamile.I-canonical R isakhi sofuzo saphinde satholakala esifundeni se-genomic siphethe indawo enkulu yokumelana ne-anthracnose ku-lupine 101 emhlophe.
Umsebenzi wethu ubonisa ukuthi ukusabela okusheshayo kokumelana, okwenziwa kusebenze ekuqaleni kokutheleleka kwezitshalo (okungcono kungabi ngemva kuka-12 hpi), kuvikela ngempumelelo i-lupine enamahlamvu amancane ku-anthracnose ebangelwa ukhunta oluyi-pathogenic i-Collelotrichum lupini.Sisebenzisa ukulandelana komthamo ophezulu, sibonise amaphrofayili enkulumo ehlukile yezakhi zofuzo zokumelana ne-anthracnose ezitshalweni ze-NLL ezixhunywe yi-Lanr1 kanye ne-AnMan resistance gene.Ukuzivikela okuphumelelayo kuhilela ukuklama ngokucophelela izakhi zofuzo zamaprotheni ahilelekile ku-redox, photosynthesis, kanye ne-pathogenesis phakathi namahora ambalwa isitshalo sithintane ne-pathogen.Ukusabela okufanayo kokuvikela, kodwa ukubambezeleka ngesikhathi, akusebenzi kahle kakhulu ekuvikeleni izitshalo ezifweni.Ukumelana ne-anthrax kulamula isakhi sofuzo se-Lanr1 kufana nokusabela okusheshayo okuvamile kofuzo luka-R (ukungavikeleki okubangelwa umphumela), kuyilapho ufuzo lwe-AnMan cishe lunikeza impendulo evundlile (ukungavikeleki okubangelwa iphethini yamangqamuzana ehlobene ne-microbe), ehlinzeka ngezinga elilingene lokusimama.
Imigqa engama-215 ye-NLL esetshenziselwa ukuhlolela omaka be-anthracnose yayihlanganisa izinhlobo ezingama-74, imigqa engama-60 etholwe ngokuwela noma ngokuzalanisa, ama-mutants angu-5, kanye nama-germplasms asendle angama-76 noma okuqala.Imigqa yayivela emazweni angu-17, ikakhulukazi ePoland (58), Spain (47), Germany (27), Australia (26), Russia (19), Belarus (7), Italy (5) neminye imigqa.emazweni ayi-10.Isethi futhi ihlanganisa imigqa ekwazi ukumelana nezithenjwa: 83A:476, Tanjil, Wonga ethwele i-Lanr1 allele, kanye ne-Mandelup ephethe i-AnMan allele.Imigqa itholwe ku-European Lupine Genetic Resource Database enakekelwa yi-Poznań Plant Breeding Ltd., Wiatrowo, Poland (Ithebula Le-Supplementary S1).
Izitshalo zazitshalwa ngaphansi kwezimo ezilawulwayo (isikhathi sesithombe amahora angu-16, izinga lokushisa libe ngu-25°C emini kanye no-18°C ebusuku).Kuhlaziywe izifaniso ezimbili zezinto eziphilayo.I-DNA yahlukaniswa emaqabunga amasonto amathathu kusetshenziswa i-DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) ngokusho kwephrothokholi.Izinga nokugxila kwe-DNA ehlukanisiwe kwahlolwa ngezindlela ze-spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA).Umaka we-AnManM1 omaka uhlobo lokumelana ne-anthracnose i-AnMan (etholakala ku-cv. Mandelup) kanye nomaka i-Anseq3 ne-Anseq4 ehlangothini lofuzo i-Lanr1 (ephuma ku-cv. Tanjil) kwahlaziywa 11,26,28.Ama-Homozygote e-allele eqinile atholwe njengokuthi “1″, okungenwa kalula kalula – njengokuthi “0”, nama-heterozygotes – njengo-0.5.
Ngokusekelwe emiphumeleni yokuhlolwa komaka i-AnManM1, i-AnSeq3 ne-AnSeq4 kanye nokuba khona kwembewu yokuhlolwa kokugcina kokulandelela, imigqa engu-50 ye-NLL ikhethelwe i-phenotyping yokumelana ne-anthracnose.Ukuhlaziywa kwenziwe ngokuphindwe kabili endlini ebamba ukushisa elawulwa ngekhompuyutha enenkathi yesithombe yamahora angu-14 enebanga lokushisa elingu-22°C phakathi no-19°C ebusuku.Imbewu iyaklwejwa (ukusika ijazi lembewu ngakolunye uhlangothi lwe-embryo nge-blade ecijile) ngaphambi kokuhlwanyela ukuvimbela ukulala kwembewu ngenxa yokuqina kwembewu kanye nokuqinisekisa ukuhluma okufanayo.Izitshalo zazitshalwa ezimbizeni (11 × 11 × 21 cm) ezinomhlaba oyinyumba (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland).Ukujova kwenziwa nge-Colletotrichum lupini Col-08 strain, eyakhula ngo-1999 kusukela eziqwini zezitshalo ze-lupine ezinamacembe amancane ezitshalwe ensimini yase-Verzhenítsa, e-Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E ).Thola indawo.Ama-isolate akhuliswe nge-SNA medium ngo-20° C. ngaphansi kokukhanya okumnyama izinsuku ezingu-21 ukuze enze i-sporulation.Emasontweni amane emva kokuhlwanyela, lapho izitshalo sezifinyelele esigabeni samaqabunga angu-4-6, ukujova kwenziwa ngokufafaza ngokumiswa kwe-conidia ekuhlanganiseni kwe-0.5 x 106 conidia nge-ml.Ngemuva kokujova, izitshalo zagcinwa ebumnyameni amahora angama-24 kumswakama ongaba ngu-98% kanye nezinga lokushisa elingu-25°C ukuze kube lula ukuhluma kwe-conidia kanye nenqubo yokutheleleka.Izitshalo zabe sezitshalwa ngaphansi kwe-photoperiod yamahora ayi-14 ku-22°C ngosuku/19°C ebusuku kanye nomswakama ongu-70%.Isilinganiso sesifo senziwa ezinsukwini ezingu-22 ngemva kokugonywa futhi sisukela ku-0 (amasosha omzimba) kuya ku-9 (okungelula kakhulu) kuye ngokuba khona noma ukungabi khona kwezilonda ze-necrotic eziqwini nasemaqabungeni.Ngaphezu kwalokho, ngemva kokufaka amagoli, isisindo sezitshalo salinganiswa.Ubudlelwano phakathi kwama-marker genotypes kanye ne-phenotypes yesifo babalwe njengephuzu lokuhlobana okulandelanayo okubili (ukungabikho komaka be-heterozygous kusethi yemigqa yokuhlaziywa kwe-phenotype yokumelana ne-anthracnose).