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Angustifolius lupine (NLL, Lupinus angustifolius L.) yog ib hom nroj tsuag leguminous uas siv rau kev tsim khoom noj thiab kev txhim kho av. Kev nthuav dav thoob ntiaj teb ntawm NLL ua qoob loo tau nyiam ntau yam kab mob fungal, suav nrog lupine anthracnose, uas ua rau muaj kab mob anthracnose loj heev. Ob lub alleles, Lanr1 thiab AnMan, uas ua rau muaj kev tiv thaiv ntau ntxiv, tau siv rau hauv kev yug tsiaj NLL, tab sis cov txheej txheem molecular tseem tsis tau paub. Hauv kev tshawb fawb no, cov cim Lanr1 thiab AnMan tau siv los tshuaj xyuas cov qauv European NLL. Kev kuaj tshuaj tiv thaiv kab mob hauv ib puag ncig tswj tau lees paub qhov ua tau zoo ntawm ob tus neeg pub dawb tiv thaiv. Kev txheeb xyuas cov noob caj noob ces sib txawv tau ua rau cov kab sawv cev tiv thaiv thiab cov kab uas yooj yim kis tau. Kev tiv thaiv Anthracnose tau cuam tshuam nrog kev tshaj tawm ntawm cov lus ontology gene "GO: 0006952 Defense Response", "GO: 0055114 Redox Process", thiab "GO: 0015979 Photosynthesis". Ntxiv rau, cov kab Lanr1 (83A: 476) tau qhia txog kev hloov pauv transcriptome sai sai tom qab txhaj tshuaj, thaum lwm cov kab tau qhia txog kev ncua sijhawm hauv qhov kev teb no li ntawm 42 teev. Cov lus teb tiv thaiv yog cuam tshuam nrog TIR-NBS, CC-NBS-LRR thiab NBS-LRR noob, 10 cov protein koom nrog hauv pathogenesis, lipid transfer proteins, endoglucan-1,3-β-glucosidase, glycine-nplua nuj cell phab ntsa proteins, thiab noob los ntawm txoj kev reactive ntawm oxygen. Cov lus teb thaum ntxov rau 83A: 476, suav nrog kev ceev faj ntawm cov noob cuam tshuam nrog photosynthesis, coincided nrog kev tiv thaiv zoo thaum lub sijhawm loj hlob ntawm cov nroj tsuag ntawm fungal biology, qhia tias tus effector ua rau muaj kev tiv thaiv kab mob. Mandeloop cov tshuaj tiv thaiv qeeb qeeb, zoo li qhov kev rub kab rov tav tag nrho.
Cov lupine nplooj nqaim (NLL, Lupinus angustifolius L.) yog cov khoom noj muaj protein ntau uas pib los ntawm thaj chaw Mediterranean sab hnub poob1,2. Tam sim no nws raug cog ua zaub mov rau tsiaj txhu thiab tib neeg. Nws kuj suav tias yog cov quav ntsuab hauv cov txheej txheem hloov pauv qoob loo vim yog kev kho nitrogen los ntawm cov kab mob symbiotic nitrogen thiab kev txhim kho tag nrho ntawm cov qauv av. NLL tau dhau los ua cov txheej txheem sai ntawm kev yug tsiaj hauv lub xyoo pua dhau los thiab tseem nyob hauv qab kev nyuaj siab ntawm kev yug me nyuam3,4,5,6,7,8,9,10,11,12. Nrog rau kev cog qoob loo dav dav ntawm NLL, kev sib law liag ntawm cov kab mob pathogenic tau tsim cov niches ua liaj ua teb tshiab thiab ua rau muaj kab mob tshiab uas ua rau cov qoob loo puas tsuaj. Qhov tshwj xeeb tshaj plaws rau cov neeg ua liaj ua teb thiab cov neeg yug tsiaj lupin yog qhov tshwm sim ntawm anthracnose, uas tshwm sim los ntawm cov pwm pathogenic, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Qhov tshwj xeeb tshaj plaws rau cov neeg ua liaj ua teb thiab cov neeg yug tsiaj lupin yog qhov tshwm sim ntawm anthracnose, uas tshwm sim los ntawm cov pwm pathogenic, Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Наиболее примечательным для фермеров и селекционеров люпина было появление антракноза, вызванного появление антракноза, вызванного появление Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Qhov tseem ceeb tshaj plaws rau cov neeg ua liaj ua teb thiab cov neeg yug tsiaj lupine yog qhov tshwm sim ntawm anthracnose los ntawm cov pwm pathogenic Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由病原真 真菌& Hagedorn13 引起的.对于羽扇豆农民和饲养者来说，最引人注目的是炭疽病的出现，它是由病原真菌lwb (Bondar) 嵵Haired.1 Наиболее поразительным для фермеров и селекционеров люпина является появление антракноза, выггываепм грибком Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13. Qhov ua rau cov neeg ua liaj ua teb thiab cov neeg yug tsiaj lupine xav tsis thoob tshaj plaws yog qhov tshwm sim ntawm anthracnose los ntawm cov pwm pathogenic Colletotrichum lupini (Bondar) Nirenberg, Feiler & Hagedorn13.Cov ntaub ntawv qhia txog tus kab mob thaum ntxov tshaj plaws tau los ntawm Brazil thiab Tebchaws Meskas, nrog rau cov tsos mob tshwm sim rau xyoo 1912 thiab 1929. Txawm li cas los xij, tom qab li 30 xyoo, tus kab mob tau raug xaiv ua 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 hauv Targeted Morphology. & H. Schrenk,. & H. Schrenk, .thiab H. Schrenk. & H.施伦克, . & H.施伦克, .thiab H. Schlenk,.Kev kuaj mob ua ntej uas tau ua nyob rau nruab nrab xyoo pua 20th tau qhia txog qee qhov kev tiv thaiv hauv NLL thiab cov lupine daj (L. luteus L.) accessions, tab sis txhua qhov lupine dawb (L. albus L.) accessions uas tau kuaj pom muaj kev phom sij heev15,16. Cov kev tshawb fawb tau qhia tias kev loj hlob ntawm anthracnose yog txuam nrog kev nce ntxiv ntawm cov nag (huab cua noo) thiab kub (hauv qhov ntau ntawm 12-28 ° C), ua rau muaj kev ua txhaum ntawm kev tiv thaiv ntawm qhov kub siab dua17, 18. Qhov tseeb, lub sijhawm xav tau rau conidia kom tawg thiab tus kab mob pib, yog plaub zaug luv dua ntawm 24 ° C (4 teev) dua li ntawm 12 ° C (16 teev) nyob rau hauv cov huab cua noo siab19. Yog li, kev sov ntawm lub ntiaj teb uas txuas ntxiv mus tau ua rau muaj kev sib kis ntawm anthracnose. Txawm li cas los xij, tus kab mob tau pom nyob rau hauv Fabkis (1982) thiab Ukraine (1983) ua tus harbinger ntawm kev hem thawj uas yuav los txog, tab sis pom tseeb tias kev lag luam lupine tsis quav ntsej thaum lub sijhawm20,21. Ob peb xyoos tom qab, tus kab mob phem no tau kis mus thoob plaws ntiaj teb thiab kuj tau cuam tshuam rau cov teb chaws loj uas tsim cov lupine xws li Australia, Poland thiab Lub Tebchaws Yelemees22,23,24. Tom qab muaj kev sib kis kab mob anthracnose hauv nruab nrab xyoo 1990, kev tshuaj xyuas ntau yam tau ua rau pom ntau tus neeg pub dawb uas tiv taus kab mob hauv cov qauv NLL19. NLL kev tiv thaiv kab mob anthracnose raug tswj los ntawm ob qho alleles tseem ceeb sib cais pom nyob rau hauv ntau qhov chaw sib txawv ntawm cov noob caj noob ces: Lanr1 hauv cultivar Tanjil thiab Wonga thiab AnMan hauv cultivar. Mandalay25,26. Cov alleles no ua tiav cov cim molecular uas txhawb nqa kev xaiv cov noob caj noob ces tiv taus kab mob hauv cov kev pab cuam yug me nyuam25,26,27,28,29,30. Cov kab mob tiv taus 83A: 476 nqa Lanr1 allele tau hla nrog cov kab qus P27255 kom tau txais cov pej xeem RIL sib cais rau kev tiv thaiv anthracnose, uas ua rau nws muaj peev xwm muab Lanr1 locus rau chromosome NLL-1131, 32, 33. Kev sib phim ntawm cov cim qhia kev sib txuas los ntawm flanking resistance loci mus rau anthracnose nrog lub moj khaum genomic, NLL tau qhia qhov chaw ntawm tag nrho peb alleles ntawm tib chromosome (NLL-11), tab sis nyob rau hauv qhov chaw sib txawv29,34,35. Txawm li cas los xij, vim muaj cov lej me me ntawm RILs thiab qhov deb ntawm cov cim thiab cov alleles sib xws, tsis muaj cov lus xaus txhim khu kev qha tuaj yeem kos txog lawv cov noob caj noob ces hauv qab. Ntawm qhov tod tes, kev siv cov noob caj noob ces rov qab hauv lupins yog qhov nyuaj vim lawv cov peev xwm rov ua dua tshiab qis heev, uas ua rau kev hloov pauv noob caj noob ces cumbersome37.
Kev tsim cov noob caj noob ces uas nqa cov allele uas xav tau hauv lub xeev homozygous, xws li 83A: 476 (Lanr1) thiab Mandelup (AnMan), tau qhib lub qhov rooj rau kev kawm txog kev tiv thaiv anthracnose thaum muaj kev sib xyaw ua ke ntawm cov alleles hauv cov pej xeem qus. Tej zaum ntawm cov txheej txheem molecular. Piv cov lus teb tiv thaiv uas tsim los ntawm cov genotypes tshwj xeeb. Kev tshawb fawb no tau soj ntsuam qhov kev teb transcriptome thaum ntxov ntawm NLL rau C. lupini kev txhaj tshuaj tiv thaiv. Ua ntej, ib lub vaj huam sib luag European NLL germplasm uas muaj 215 kab tau tshuaj xyuas siv cov cim molecular uas cim Lanr1 thiab AnMan alleles. Anthracnose phenotyping tau ua tiav rau 50 NLL kab, yav dhau los xaiv rau cov cim molecular, nyob rau hauv cov xwm txheej tswj hwm. Raws li cov kev sim no, plaub kab sib txawv hauv kev tiv thaiv anthracnose thiab Lanr1 / AnMan allelic composition tau xaiv rau kev tiv thaiv cov noob caj noob ces sib txawv siv ob txoj hauv kev sib txuas: high-throughput RNA sequencing thiab real-time PCR quantification.
Kev tshuaj ntsuam ntawm ib pawg ntawm NLL germplasm (N = 215) nrog cov cim Lanr1 (Anseq3 thiab Anseq4) thiab AnMan (Anseq4) thiab AnMan (AnManM1) qhia tau hais tias tsuas yog ib kab (95726, ze Salamanca-b) ua kom loj dua qhov "tsis kam" allele rau txhua tus cim, thaum "Muaj cov alleles 'susceptible'" pom qhov sib piv ntawm txhua tus cim hauv 158 (~ 73.5%) kab. Kaum peb kab tsim ob lub "tsis kam" alleles ntawm Lanr1 cim, thiab 8 kab tsim cov "tsis kam" alleles ntawm Lanr1. cim. Lub "tsis kam" allele ntawm AnMan cim (Supplementary Table S1). Ob kab yog heterozygous rau Anseq3 cim thiab ib qho heterozygous rau AnManM1 cim. 42 kab (19.5%) nqa cov theem sib txawv ntawm Anseq3 thiab Anseq4 alleles, qhia txog qhov muaj ntau zaus ntawm kev sib xyaw ua ke ntawm ob lub loci no. Cov yam ntxwv ntawm Anthracnose nyob rau hauv cov xwm txheej tswj hwm (Supplementary Table S2) qhia txog kev hloov pauv ntawm kev tiv thaiv ntawm cov genotypes kuaj, uas tau pom nyob rau hauv qhov hnyav ntawm anthracnose. Qhov sib txawv ntawm cov qhab nia nruab nrab yog txij li 1.8 (tiv taus nruab nrab) txog 6.9 (ua rau muaj kev phom sij) thiab qhov hnyav ntawm cov nroj tsuag sib txawv yog txij li 0.62 (ua rau muaj kev phom sij) txog 4.45 g (ua rau muaj kev phom sij). Muaj kev sib raug zoo tseem ceeb ntawm cov nqi pom hauv ob qhov kev rov ua dua ntawm qhov kev sim (0.51 rau cov qhab nia hnyav ntawm kab mob, P = 0.00017 thiab 0.61 rau qhov hnyav ntawm cov nroj tsuag, P < 0.0001) nrog rau ntawm ob qho kev ntsuas no (- 0.59 thiab - 0.77, P < 0.0001). Muaj kev sib raug zoo tseem ceeb ntawm cov nqi pom hauv ob qhov kev rov ua dua ntawm qhov kev sim (0.51 rau cov qhab nia mob hnyav, P = 0.00017 thiab 0.61 rau qhov hnyav ntawm cov nroj tsuag, P <0.0001) nrog rau ntawm ob qho kev ntsuas no (- 0.59 thiab - 0.77, P <0.0001). Выявлена ​​достоверная корреляция между значениями, наблюдаемыми в двух повторностях эксперовентлежтал (0,5 болезни, P = 0,00017 thiab 0,61 для массы растения, P < 0,0001), а также между этими двумя параметрами 7,0,019 (-0,59) 0,0001). Muaj kev sib raug zoo tseem ceeb ntawm cov nqi pom hauv ob qhov kev rov ua dua ntawm kev sim (0.51 rau cov qhab nia mob hnyav, P = 0.00017 thiab 0.61 rau qhov hnyav ntawm cov nroj tsuag, P <0.0001), nrog rau ntawm ob qho kev ntsuas no (- 0.59 thiab -0.77, P <0.0001) 0.0001).在两次重复实验中观察到的值之间存在显着相关性（疾病严重程度评分为0.51，P = 0.00017，植物重量为0.61，P < 0.0001）以及这两个参数之间（- 0.59 和- 0.77，P < 0.0001).在两次重复实验中观察的值 之间存在相关性（疾病严重程度评分0亦评分0。。。。 p = 0.00017 ， 植物 为 0.61 ， p < 0.0001) 以及 两 个参数 之间 （（（- 0.59 和– 0.59 . 9 . 0.77, P <0.0001)。 Наблюдалась значительная корреляция между значениями, наблюдаемыми в двух повторностях (оценка тя, 0 зажест) 0,00017 и масса растения 0,61, P <0,0001), thiab между этими двумя параметрами (-0,59 и -0,0001) 0,77, P <0.10, Muaj kev sib raug zoo tseem ceeb ntawm cov nqi pom hauv cov lej sib npaug (qhov mob hnyav ntawm tus kab mob 0.51, P = 0.00017 thiab qhov hnyav ntawm cov nroj tsuag 0.61, P <0.0001) thiab ntawm ob qho kev ntsuas no (-0.59 thiab -0.0001) 0.77, P<0.0001. ).Cov tsos mob feem ntau pom hauv cov nroj tsuag uas muaj kev phom sij suav nrog kev khoov thiab ntswj ntawm cov qia zoo li tus qauv "yeeb ncuab tus tswv yug yaj", ua raws li cov qhov txhab oval nrog cov txiv kab ntxwv / liab sporozoites (Daim Duab Ntxiv 1). Cov khoom siv Australian uas nqa cov noob Lanr1 (83A: 476 thiab Tanjil) thiab AnMan (Mandelup) muaj kev tiv thaiv nruab nrab, 0.0331 thiab 0.0036). Qee cov kab uas kuj nqa cov alleles Lanr1 thiab / lossis AnMan "tiv taus" qhia cov tsos mob ntawm tus kab mob.
Qhov nthuav yog, ob peb kab NLL uas tsis muaj cov cim "resistant" allele tau qhia txog qib siab ntawm kev tiv thaiv anthracnose (piv lossis siab dua rau Lanr1 lossis AnMan genotypes), xws li Boregine (P tus nqi < 0.0001 rau ob qho tib si), Bojar (P tus nqi < 0.0001 rau qhov qhab nia thiab 0.001 rau qhov hnyav ntawm tsob ntoo) thiab Population B-549/79b (P tus nqi < 0.0001 rau qhov qhab nia thiab tsis tseem ceeb rau qhov hnyav). Qhov nthuav yog, ob peb kab NLL uas tsis muaj cov cim "resistant" allele tau qhia txog qib siab ntawm kev tiv thaiv anthracnose (piv lossis siab dua rau Lanr1 lossis AnMan genotypes), xws li Boregine (P tus nqi < 0.0001 rau ob qho tib si), Bojar (P tus nqi < 0.0001 rau qhov qhab nia thiab 0.001 rau qhov hnyav ntawm tsob ntoo) thiab Population B-549/79b (P tus nqi < 0.0001 rau qhov qhab nia thiab tsis tseem ceeb rau qhov hnyav). Интересно, что несколько линий NLL, лишенных какого-либо «резистентного» маркерного аллеля, показьрали устойчивости к антракнозу (сопоставимый или более высокий, чем для генотипов Lanr1 или AnMan), таких как 1чани0де ( обоих параметров), Bojar (значение P < 0,0001 для оценки и 0,001 для массы растения) и популяции B-549/79b (значение P <0,0001 для оценки и незначимо для массы). Qhov nthuav yog, ntau kab NLL uas tsis muaj cov cim 'resistant' allele tau qhia txog qib siab ntawm kev tiv thaiv rau anthracnose (piv rau lossis siab dua rau Lanr1 lossis AnMan genotypes), xws li Boregine (P tus nqi < 0.0001 rau ob qho tib si), Bojar (P tus nqi < 0.0001 rau kev ntsuam xyuas thiab 0.001 rau qhov hnyav ntawm cov nroj tsuag) thiab cov pej xeem B-549/79b (P tus nqi < 0.0001 rau kev ntsuam xyuas thiab tsis tseem ceeb rau qhov hnyav).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斧Man (Lan 1 与基因型相当或更高), 例如Boregine (两个参数的P 值< 0.0001), Bojar (P 值<得分为0.0001，植物重量为0.001）和种群B-549/79b (得分P 值< 0.0001，重量不显着). Nws yog qhov nthuav tias qee lub tshuab NLL uas tsis muaj cov cim "antigenic" qhia txog kev tiv thaiv kab rov tav siab (sib npaug rau Lanr1 lossis AnMan noob lossis siab dua), xws li Boregine (ob qho tib si P < 0.0001), Bojar (P tus nqi < 0.0001, qhov hnyav ntawm tsob nroj 0.001) thiab hom B-549/79b (P tus nqi < 0.0001, qhov hnyav tsis tseem ceeb). Интересно, что некоторые линии NLL, лишенные каких-либо маркерных аллелей «резистентности», показалини высти устойчивости к антракнозу (сравнимые или выше, чем у генотипов Lanr1 или AnMan), такие как Boregine (значение P <дратение P <дратобом) Bojar (значение P <0,0001, масса растения 0,001) и популяция B-549/79b (оценка P-значение <0,0001, масса незначительна). Qhov txaus nyiam yog, qee cov kab NLL uas tsis muaj cov cim 'tiv taus' alleles qhia tau tias muaj kev tiv thaiv anthracnose ntau (piv rau lossis siab dua Lanr1 lossis AnMan genotypes), xws li Boregine (P tus nqi rau ob qho tib si <0.0001), Bojar (P-tus nqi <0.0001, qhov hnyav ntawm tsob ntoo 0.001) thiab cov pejxeem B-549/79b (P-tus nqi <0.0001, qhov hnyav tsis tseem ceeb).Qhov xwm txheej no qhia txog qhov ua tau ntawm qhov tshiab ntawm cov noob caj noob ces ntawm kev tiv thaiv, piav qhia txog qhov tsis muaj kev sib raug zoo ntawm cov cim genotypes thiab cov kab mob phenotypes (P tus nqi ntawm ~ 0.42 txog ~ 0.98). Yog li, Kolmogorov-Smirnov xeem tau qhia tias cov ntaub ntawv ntawm kev tiv thaiv anthracnose tau kwv yees li ib txwm faib rau cov qhab nia (P-tus nqi 0.25 thiab 0.11) thiab cov nroj tsuag loj (P-tus nqi 0.47 thiab 0.55), qhia tias kuv xav tias ntau alleles dua li Lanr1 thiab AnMan koom nrog.
Raws li cov txiaj ntsig ntawm kev tshuaj ntsuam xyuas kev tiv thaiv anthracnose, 4 kab tau raug xaiv rau kev tshuaj xyuas transcriptome: 83A: 476, Boregine, Mandelup, thiab Population 22660. Cov kab no tau rov sim dua rau kev tiv thaiv anthrax hauv kev sim inoculation los ntawm RNA sequencing, muab tias lawv zoo ib yam li hauv kev xeem dhau los. Cov qhab nia yog raws li nram no: Boregin (1.71 ± 1.39), 83A: 476 (2.09 ± 1.38), Mandelup (3.82 ± 1.42) thiab cov pej xeem 22660 (6.11 ± 1.29).
Cov txheej txheem Illumina NovaSeq 6000 tau ua tiav qhov nruab nrab ntawm 40.5 Mread khub ib qho qauv (29.7 txog 54.4 Mreads) (Supplementary Table S3). Cov qhab nia sib phim hauv cov kab ke siv tau txij li 75.5% txog 88.6%. Qhov nruab nrab ntawm kev sib raug zoo ntawm cov ntaub ntawv suav nyeem ntawm cov kev hloov pauv ntawm kev sim ntawm cov kab mob rov ua dua tau txij li 0.812 txog 0.997 (qhov nruab nrab 0.959). Tawm ntawm 35,170 cov noob caj noob ces uas tau soj ntsuam, 2917 tsis tau qhia txog kev qhia tawm, thiab lwm 4785 cov noob caj noob ces tau qhia tawm ntawm qib tsis tseem ceeb (qhov nruab nrab < 5). Tawm ntawm 35,170 cov noob caj noob ces uas tau soj ntsuam, 2917 tsis tau qhia txog kev qhia tawm, thiab lwm 4785 cov noob caj noob ces tau qhia tawm ntawm qib tsis tseem ceeb (qhov nruab nrab < 5). Из 35 170 проанализированных генов 2917 не проявляли экспрессии, а остальные 4785 генов экспрельсир незначительном уровне (базовое среднее <5). Ntawm 35,170 cov noob caj noob ces uas tau soj ntsuam, 2917 tsis qhia txog kev qhia tawm, thiab cov noob caj noob ces seem 4785 tau qhia tawm ntawm qib tsis tseem ceeb (qhov nruab nrab ntawm lub hauv paus <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（幇最）35,170 Из 35 170 проанализированных генов 2917 не экспрессировались, а остальные 4785 генов имели незкьсуют (bазовое среднее значение <5). Ntawm 35,170 cov noob caj noob ces uas tau soj ntsuam, 2917 tsis tau qhia tawm thiab cov noob caj noob ces seem 4785 muaj kev qhia tawm tsis tseem ceeb (qhov nruab nrab ntawm lub hauv paus <5).Yog li, tus naj npawb ntawm cov noob caj noob ces uas tau suav tias tau qhia tawm (qhov nruab nrab ≥ 5) thaum lub sijhawm sim yog 27,468 (78.1%) (Supplementary Table S4).
Txij thawj lub sijhawm, txhua kab NLL tau teb rau kev txhaj tshuaj ntawm C. lupini (strain Col-08) los ntawm kev rov ua dua qhov transcriptome (Rooj 1), txawm li cas los xij, qhov sib txawv tseem ceeb tau pom ntawm cov kab. Yog li, kab tiv thaiv 83A: 476 (nqa lub Lanr1 gene) tau qhia txog kev rov ua dua qhov transcriptome tseem ceeb ntawm thawj lub sijhawm (6 hpi) nrog 31-69-npaug nce ntxiv hauv tus lej ntawm cov noob caj noob ces cais tawm piv rau lwm lub sijhawm ntawm lub sijhawm no. Tsis tas li ntawd, qhov siab tshaj plaws no yog luv luv, vim tias kev qhia tawm ntawm tsuas yog ob peb lub noob caj noob ces tseem hloov pauv tseem ceeb ntawm lub sijhawm thib ob (12 hpi). Qhov nthuav yog, Boregine, uas kuj tau qhia txog qib siab ntawm kev tiv thaiv hauv kev sim graft, tsis tau dhau los ua qhov kev hloov pauv loj heev thaum lub sijhawm sim. Txawm li cas los xij, tus lej ntawm cov noob caj noob ces qhia tawm sib txawv (DEG) yog tib yam rau Boregine thiab 83A: 476 ntawm 12 HPI. Ob leeg Mandelup thiab cov pej xeem 22660 tau qhia txog DEG peaks ntawm lub sijhawm kawg (48 l / s), qhia txog kev ncua sijhawm hauv kev tiv thaiv teb.
Vim tias 83A: 476 tau dhau los ua qhov kev hloov pauv loj heev los teb rau C. lupini ntawm 6 HPI piv rau txhua kab, ~ 91% ntawm DEGs uas tau pom ntawm lub sijhawm no yog cov kab mob tshwj xeeb (Daim Duab 1). Txawm li cas los xij, muaj qee qhov kev sib tshooj hauv cov lus teb thaum ntxov ntawm cov kab kawm, raws li 68.5%, 50.9%, thiab 52.6% DEG hauv Boregine, Mandelup, thiab cov pej xeem 22660, feem, sib tshooj nrog cov uas pom hauv 83A: 476 hauv qee lub sijhawm. Txawm li cas los xij, cov DEGs no tsuas yog ib feem me me (0.97–1.70%) ntawm tag nrho cov DEGs uas tam sim no pom siv 83A: 476. Ntxiv rau, 11 DEGs los ntawm txhua kab tau sib xws thaum lub sijhawm no (Cov Lus Qhia Ntxiv S4-S6), suav nrog cov khoom sib xws ntawm cov lus teb tiv thaiv cog qoob loo: lipid transfer protein (TanjilG_32225), endoglucan-1,3-β-glucoside enzyme (TanjilG_23384), ob qho protein uas ua rau muaj kev ntxhov siab zoo li SAM22 (TanjilG_31528 thiab TanjilG_31531), cov protein latex yooj yim (TanjilG_32352), thiab ob qho protein glycine-nplua nuj cell phab ntsa protein (TanjilG_19701 thiab TanjilG_19702). Kuj tseem muaj qhov sib tshooj siab hauv cov lus teb transcriptome ntawm 83A: 476 thiab Boregine ntawm 24 HPI (tag nrho 16-38% DEG) thiab ntawm Mandelup thiab Population 22660 ntawm 48 HPI (tag nrho 14-20% DEG).
Daim duab Venn qhia txog tus lej ntawm cov noob caj noob ces sib txawv (DEG) hauv cov kab lupine nqaim (NLL) uas tau txhaj tshuaj nrog Colletotrichum lupini (hom Col-08 tau los ntawm cov teb lupine hauv Wierzhenice, Poland, 1999). Cov kab NLL uas tau tshuaj xyuas yog: 83A: 476 (tiv taus, nqa Lanr1 allele), Boregine (tiv taus, keeb kwm yav dhau los tsis paub), Mandelup (tiv taus nruab nrab, nqa AnMan allele) thiab pej xeem 22660 (muaj kev phom sij heev). Lub npe luv hpi sawv cev rau ntau teev tom qab txhaj tshuaj. Cov nqi xoom tau raug tshem tawm los ua kom daim duab yooj yim dua.
Cov noob caj noob ces uas tau tshaj tawm ntau dhau ntawm 6 hpi tau raug tshuaj xyuas seb puas muaj cov noob caj noob ces R uas tau teev tseg (Supplementary Table S7). Kev tshawb fawb no tau qhia txog kev hloov pauv ntawm cov noob caj noob ces uas tiv taus kab mob nrog cov noob caj noob ces NBS-LRR tsuas yog ntawm 83A: 476. Cov noob caj noob ces no muaj ib qho noob caj noob ces TIR-NBS-LRR (tanjilg_05042), tsib lub noob caj noob ces CC-NBS-LRR (tanjilg_06165, tanjilg_06162, tanjilg_22773, tanjilg_22640, thiab tanjilg_16162), thiab Plaub NBS-LR, Tanjilg_16162), thiab Plaub NBS-LRRE (tanjilg_16162) nrog rau Plaub NBS-Lrr (tanjilg_16162) thiab Plaub NBS-LRR (TANJILG_16162). Tag nrho cov noob caj noob ces no muaj cov noob caj noob ces uas tau teeb tsa hauv cov kab ke uas tau khaws cia. Ntxiv rau cov noob caj noob ces NBS-LRR, ntau lub RLL kinases tau qhib ntawm 6 hpi, uas yog ib qho hauv Boregine (TanjilG_19877), ob qho hauv Mandelup (TanjilG_07141 thiab TanjilG_19877) thiab hauv cov pej xeem 22660 (TanjilG_09014 thiab TanjilG_10361) thiab ob qho hauv 83A 27:476.
Cov noob caj noob ces uas muaj kev hloov pauv ntau yam hauv kev teb rau kev txhaj tshuaj nrog C. lupini (strain Col-08) tau raug rau Gene Ontology (GO) kev tshuaj xyuas kev nplua nuj (Supplementary Table S8). Cov lus hais txog cov txheej txheem ntawm lub cev uas feem ntau hais ntau dhau yog "GO: 0006952 kev tiv thaiv" uas tau tshwm sim hauv 6 ntawm 16 (lub sijhawm × kab) kev sib xyaw ua ke nrog qhov tseem ceeb siab (P tus nqi < 0.001) (Daim duab 2). Cov lus hais txog cov txheej txheem ntawm lub cev uas feem ntau hais ntau dhau yog "GO: 0006952 kev tiv thaiv" uas tau tshwm sim hauv 6 ntawm 16 (lub sijhawm × kab) kev sib xyaw ua ke nrog qhov tseem ceeb siab (P tus nqi < 0.001) (Daim duab 2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 зыйкитный, появлялся в 6 из 16 (время × линия) комбинаций с высокой значимостью (значение P <0,001) (рис. 2). Cov lus hais txog cov txheej txheem ntawm lub cev uas feem ntau hais ntau dhau yog 'GO:0006952 kev tiv thaiv', uas tau tshwm sim hauv 6 ntawm 16 (lub sijhawm × kab lis kev cai) kev sib xyaw ua ke nrog qhov tseem ceeb siab (P tus nqi < 0.001) (Daim duab 2).最常被过度代表的生物过程术语是“GO: 0006952 防御反应”，它出的现在16 个（时痴×琿）个中，具有高显着性（P 值< 0.001）（图2）. Cov lus piav qhia txog cov txheej txheem ntawm lub cev uas sawv cev tshaj plaws yog "GO:0006952 kev tiv thaiv", uas tshwm sim hauv 6 ntawm 16 (时间 × 线) kev sib xyaw ua ke, nrog rau qhov tseem ceeb siab (P tus nqi < 0.001) (图2). Наиболее часто чрезмерно представленным термином биологического процесса был «GO: 0006952 Defense Response», из 16 комбинаций (время × линия) с высокой значимостью (значение P <0,001) (рис. 2). Cov lus hais txog cov txheej txheem ntawm lub cev uas feem ntau hais ntau dhau yog 'GO:0006952 Defense Response', uas tau tshwm sim hauv 6 ntawm 16 qhov kev sib xyaw ua ke (lub sijhawm × kab) nrog qhov tseem ceeb siab (P tus nqi < 0.001) (Daim duab 2).Lo lus no tau raug sawv cev ntau dhau ntawm ob lub sijhawm hauv 83A: 476 thiab Boregine (6 thiab 24 hpi) thiab ntawm ib lub sijhawm hauv Mandelup thiab Population 22660 (12 thiab 6 hpi, feem). Qhov no yog qhov tshwm sim xav tau, qhia txog kev teb antifungal ntawm cov kab tiv taus. Tsis tas li ntawd, 83A: 476 teb rau C. lupini los ntawm kev ua kom sai sai cov noob caj noob ces cuam tshuam nrog oxidative burst uas sawv cev los ntawm lo lus "GO: 0055114 redox txheej txheem", qhia txog kev teb tiv thaiv tshwj xeeb, thaum Boregine qhia txog kev teb tiv thaiv tshwj xeeb, cuam tshuam nrog lo lus 'GO'. :0006950 Kev teb rau kev ntxhov siab”. Cov pej xeem 22660 tau qhib qhov kev teb tiv thaiv kab rov tav uas muaj cov metabolites theem nrab, qhia txog ntau dhau ntawm cov lus "GO:0016104 Txheej Txheem ntawm triterpene biosynthesis" thiab "GO:0006722 Txheej Txheem ntawm triterpene metabolism" (ob lo lus koom nrog tib pawg ntawm cov noob), coj mus rau hauv tus account cov txiaj ntsig ntawm GO lub sijhawm kev tshuaj xyuas kev nplua nuj, Mandelup reaction stability yog ntawm Boregine thiab Population 22660. Tsis tas li ntawd, thaum ntxov reaction 83A:476 (6 hpi) thiab qeeb reaction Mandelup thiab Population 22660 suav nrog lo lus GO:0015979 'photosynthesis' thiab lwm yam kev ua haujlwm ntawm lub cev.
Cov lus hais txog bioprocess gene ontology xaiv hauv cov lus piav qhia ntawm cov noob qhia sib txawv thaum lub sijhawm teb transcriptome ntawm narrow-leaved lupine (NLL) uas tau txhaj tshuaj nrog anthrax lupine (Col-08 hom tau los ntawm cov teb lupine hauv Wierzhenice, Poland, xyoo 1999) yog qhov ua kom pom tseeb heev. Cov kab NLL uas tau soj ntsuam yog: 83A: 476 (tiv taus, nqa homozygous Lanr1 allele), Boregine (tiv taus, tsis paub keeb kwm yav dhau los), Mandelup (tiv taus nruab nrab, nqa homozygous AnMan allele) thiab pej xeem 22660 (ua rau muaj kev phom sij).
Vim tias txoj kev tshawb fawb no tsom mus rau kev txheeb xyuas cov noob caj noob ces uas ua rau muaj kev tiv thaiv anthracnose, cov noob caj noob ces uas tau muab rau cov lus GO "GO: 0006952 Cov lus teb tiv thaiv" thiab "GO: 0055114 Redox processes" tau raug tshuaj xyuas nrog kev txiav tawm txij li thaum qhov nruab nrab txhais tau tias ≥ 30 nrog tsawg kawg ib kab. × taw tes hauv lub sijhawm sib xyaw cov nqi tseem ceeb ntawm log2 (fold hloov pauv). Tus lej ntawm cov noob caj noob ces ua tau raws li cov qauv no yog 65 rau GO: 0006952 thiab 524 rau GO: 0055114.
83A:476 tau qhia ob lub ncov DEG uas tau sau tseg nrog lo lus GO:0006952, thawj zaug ntawm 6 noob ib nti (64 noob, kev tswj hwm nce thiab nqis) thiab qhov thib ob ntawm 24 noob ib nti (15 noob, kev tswj hwm nce xwb). Boregine kuj tau qhia tias GO:0006952 tau nce siab tshaj plaws tib lub sijhawm, tab sis nrog DEG tsawg dua (11 thiab 8) thiab kev ua haujlwm tshwj xeeb. Mandeloop tau qhia ob lub ncov ntawm GO:0006952 ntawm 12 thiab 48 HPI, ob qho tib si nqa 12 noob (thawj zaug nrog cov noob ua haujlwm, thiab qhov thib ob nrog tsuas yog cov noob suppressive), thaum cov pej xeem 22660 ntawm 6 HPI (13 noob) muaj qhov tseem ceeb dua ntawm qhov nce siab. Yuav tsum nco ntsoov tias 96.4% ntawm GO:0006952 DEG hauv cov ncov no muaj tib hom kev teb (nce lossis nqis), qhia txog kev sib tshooj tseem ceeb hauv kev tiv thaiv teb txawm tias muaj qhov sib txawv ntawm cov noob koom nrog. Pawg loj tshaj plaws ntawm cov kab ke uas cuam tshuam nrog lo lus GO: 0006952 encodes Starvation Stress-Associated Message Protein 22 (SAM22-zoo li), uas yog pawg 10 pathogenesis-associated protein (PR-10) protein clade thiab cov protein core latex. zoo sib xws (MLP-zoo li) protein (Daim Duab 3). Ob pawg sib txawv hauv qhov xwm txheej ntawm kev qhia tawm thiab kev coj ntawm kev teb. Cov noob encoding SAM22-zoo li cov proteins tau qhia txog kev sib xws thiab tseem ceeb ntawm cov ntsiab lus thaum ntxov (6 lossis 12 hpi) thiab feem ntau tsis teb thaum kawg ntawm kev sim (48 hpi), thaum MLP-zoo li cov proteins tau qhia txog kev sib koom tes ntawm 6 hpi. hpi. 83A: 476 thiab Mandelup ntawm 48 hp / hauv, yuav luag txhua qhov chaw ntawm cov ntaub ntawv tsis teb. Tsis tas li ntawd, qhov sib txawv ntawm cov qauv qhia tawm ntawm SAM22-zoo li cov protein noob tau ua raws li kev hloov pauv pom hauv kev tiv thaiv anthracnose, vim tias cov kab tiv taus ntau dua muaj ntau lub sijhawm tseem ceeb ua rau cov noob no ntau dua li cov noob uas yooj yim dua. Lwm tus noob caj noob ces LlR18A/B zoo li PR-10 tau qhia txog tus qauv qhia tawm zoo sib xws rau cov noob caj noob ces protein zoo li SAM22.
Cov khoom tseem ceeb ntawm cov txheej txheem biological lo lus "GO: 0006952 Kev Tiv Thaiv Kev Tiv Thaiv" thiab cov qauv qhia tawm ntawm cov noob caj noob ces ntawm Lanr1 thiab AnMan alleles tau txheeb xyuas. Lub Log2 nplai sawv cev rau log2 tus nqi ​​(fold hloov pauv) ntawm inoculated (Colletotrichum lupini, hom Col-08, tau txais los ntawm lupine teb, Wizhenica, Poland, 1999) thiab tswj (sham-inoculated) cov nroj tsuag tib lub sijhawm. Cov kab lupine narrowleaf hauv qab no tau tshuaj xyuas: 83A: 476 (resistant, nqa homozygous Lanr1 allele), Boregine (resistant, keeb kwm yav dhau los tsis paub), Mandelup (resistant moderately, nqa homozygous AnMan allele), thiab Population 22660 (sensitive).
Ntxiv rau, cov qauv qhia tawm ntawm RNA-seq cov noob Lanr1 (TanjilG_05042) thiab AnMan (TanjilG_12861) tau raug soj ntsuam (Daim Duab 3). Lub noob TanjilG_05042 tau qhia txog kev teb tseem ceeb (kev ua kom muaj zog) ntawm 83A: 476 tsuas yog ntawm thawj lub sijhawm (6 hpi), thaum TanjilG_12861 tseem ceeb hauv Mandeloop tsuas yog ntawm ob lub sijhawm: 6 hpi (kev tswj hwm qis) thiab 24 hpi (6 hpi). Nrog.). kho tau)).
Cov noob caj noob ces uas tau hais ntau tshaj plaws hauv lo lus GO: 0055114 "txheej txheem redox" yog cov noob caj noob ces uas encodes cytochrome P450 proteins thiab peroxidase (Daim Duab 4). Rau cov qauv cais los ntawm 83A: 476 ntawm 6 HPI, qhov siab tshaj plaws lossis tsawg kawg nkaus log2 (fold change) tus nqi (rau 86.6% ntawm cov noob caj noob ces) feem ntau tau pom ntawm cov nroj tsuag inoculated thiab cov nroj tsuag tswj, qhia txog qhov kev teb siab ntawm cov noob caj noob ces no rau kev sib deev inoculating. 83A: 476 tau qhia txog qhov tseem ceeb tshaj plaws GO: 0055114 DEG ntawm 6 hpi (503 noob caj noob ces), thaum cov kab seem ntawm 48 hpi (Boregine, 31 noob caj noob ces; Mandelup, 85 noob caj noob ces; thiab Population 22660, 78 noob caj noob ces)). Hauv feem ntau cov noob caj noob ces ntawm GO: 0055114 tsev neeg, ob hom kev teb rau kev txhaj tshuaj (kev ua kom muaj zog thiab kev txwv tsis pub muaj zog) tau pom. Txaus siab kawg li, txog li 97.6% ntawm DEGs tau txheeb xyuas rau lo lus GO: 0055114 hauv Mandelupe ntawm 48 hp Cov kev soj ntsuam no qhia tias, txawm tias qhov ntsuas me dua (piv txwv li, tus lej ntawm cov noob hloov pauv redox, 85 piv rau 503), tus qauv ntawm cov lus teb qeeb ntawm mandeloup rau anthracnose zoo ib yam li cov lus teb thaum ntxov ntawm 83A: 476. Hauv Boregine thiab Population 22660, qhov kev sib sau ua ke no qis dua ntawm 51.6% thiab 75.6%, feem.
Cov qauv ntawm kev qhia tawm ntawm cov khoom tseem ceeb ntawm lub sijhawm ntawm cov txheej txheem biological "GO: 0055114 Redox txheej txheem" ​​tau qhia tawm. Lub Log2 nplai sawv cev rau log2 tus nqi ​​(fold hloov pauv) ntawm inoculated (Colletotrichum lupini, hom Col-08, tau txais los ntawm lupine teb, Wizhenica, Poland, 1999) thiab tswj (sham-inoculated) cov nroj tsuag tib lub sijhawm. Cov kab lupine narrowleaf hauv qab no tau tshuaj xyuas: 83A: 476 (resistant, nqa homozygous Lanr1 allele), Boregine (resistant, keeb kwm yav dhau los tsis paub), Mandelup (resistant moderately, nqa homozygous AnMan allele), thiab Population 22660 (sensitive).
83A:476 Cov lus teb Transcriptomic rau kev txhaj tshuaj nrog C. lupini (strain Col-08) kuj suav nrog kev sib koom ua ke ntawm cov noob caj noob ces uas tau muab rau lo lus GO:0015979 "photosynthesis" thiab lwm yam txheej txheem ntsig txog biological (FIG. 5). Cov GO:0015979 DEG teeb tsa no muaj 105 noob caj noob ces uas tau raug txo qis ntawm 6 hpi ntawm 83A:476. Hauv pawg no, 37 noob caj noob ces kuj tau downregulated hauv Mandelup ntawm 48 HPI thiab 35 tib lub sijhawm hauv 22660 pej xeem, suav nrog 19 DEGs sib xws rau ob qho tib si genotypes. Tsis muaj DEGs cuam tshuam nrog lo lus GO: 0015979 tau ua haujlwm tseem ceeb hauv txhua qhov kev sib xyaw ua ke (kab x lub sijhawm).
Cov qauv ntawm kev qhia tawm ntawm cov khoom tseem ceeb ntawm lub sijhawm ntawm cov txheej txheem biological "GO: 0015979 Photosynthesis" tau qhia tawm. Lub Log2 nplai sawv cev rau log2 tus nqi ​​(fold hloov pauv) ntawm inoculated (Colletotrichum lupini, hom Col-08, tau txais los ntawm lupine teb, Wizhenica, Poland, 1999) thiab tswj (sham-inoculated) cov nroj tsuag tib lub sijhawm. Cov kab lupine narrowleaf hauv qab no tau tshuaj xyuas: 83A: 476 (resistant, nqa homozygous Lanr1 allele), Boregine (resistant, keeb kwm yav dhau los tsis paub), Mandelup (resistant moderately, nqa homozygous AnMan allele), thiab Population 22660 (sensitive).
Raws li cov txiaj ntsig ntawm kev tshuaj xyuas kev qhia sib txawv thiab tej zaum koom nrog kev tiv thaiv cov lus teb tiv thaiv cov kab mob pathogenic fungi, cov txheej txheem xya no tau raug xaiv rau kev ntsuas cov qauv qhia los ntawm PCR tiag tiag (Supplementary Table S9).
Cov noob protein TanjilG_10657 tau raug tsim los ntawm txhua kab thiab lub sijhawm uas tau kawm piv rau cov nroj tsuag tswj (mimic) (Cov Lus Qhia Ntxiv S10, S11). Tsis tas li ntawd, cov qauv qhia tawm ntawm TanjilG_10657 tau qhia txog kev nce ntxiv ntawm kev sim rau txhua kab. Cov pej xeem 22660 tau qhia txog kev nkag siab siab tshaj plaws ntawm TanjilG_10657 rau kev txhaj tshuaj nrog 114-fold activation thiab qib kev qhia tawm siab tshaj plaws (4.4 ± 0.4) ntawm 24 HPI (Daim Duab 6a). Cov noob protein PR10 LlR18A TanjilG_27015 kuj tau qhia txog kev ua haujlwm thoob plaws txhua kab thiab lub sijhawm, nrog rau qhov tseem ceeb ntawm cov ntaub ntawv feem ntau (Daim Duab 6b). Zoo ib yam li TanjilG_10657, qib siab tshaj plaws ntawm TanjilG_27015 tau pom nyob rau hauv 22660 cov pej xeem uas tau txhaj tshuaj ntawm 24 HPI (19.5 ± 2.4). Cov kua qaub endochitinase gene TanjilG_04706 tau nce siab heev hauv txhua kab thiab txhua lub sijhawm tshwj tsis yog Boregine 6 hpi (Daim Duab 6c). Nws tau raug tsim tawm ntawm thawj lub sijhawm (6 HPI) ntawm 83A: 476 (los ntawm 10.5 zaug) thiab nce me ntsis hauv lwm kab (los ntawm 6.6-7.5 zaug). Thaum lub sijhawm sim, qhov kev qhia tawm ntawm TanjilG_04706 tseem nyob ntawm cov qib zoo sib xws hauv 83A: 476 thiab Boregine, thaum nyob hauv Mandelup thiab Population 22660 nws tau nce siab heev, ncav cuag cov nqi siab (5.9 ± 1.5 thiab 6.2 ± 1.5, feem). Tus endoglucan-1,3-β-glucosidase-zoo li gene TanjilG_23384 tau qhia txog kev ua haujlwm siab ntawm thawj ob lub sijhawm (6 thiab 12 hpi) hauv txhua kab tshwj tsis yog cov pej xeem 22660 (Daim duab 6d). Cov qib kev qhia tawm siab tshaj plaws ntawm TanjilG_23384 tau pom ntawm lub sijhawm thib ob (12 hpi) hauv Mandelup (2.7 ± 0.3) thiab 83A: 476 (1.5 ± 0.1). Ntawm 24 HPI, TanjilG_23384 kev qhia tawm qis dua hauv txhua kab kawm (los ntawm 0.04 ± 0.009 txog 0.44 ± 0.12).
Cov qauv qhia tawm ntawm cov noob caj noob ces xaiv (ag) tau qhia los ntawm kev ntsuas PCR. Cov lej 6, 12 thiab 24 sawv cev rau ob peb teev tom qab txhaj tshuaj tiv thaiv kab mob. Cov noob caj noob ces LanDExH7 thiab LanTUB6 tau siv rau kev ua kom zoo li qub thiab LanTUB6 tau siv rau kev ntsuas sib txawv. Cov kab yuam kev sawv cev rau qhov sib txawv ntawm tus qauv raws li peb qhov kev rov ua dua ntawm cov kab mob, txhua qhov yog qhov nruab nrab ntawm peb qhov kev rov ua dua ntawm kev siv tshuab. Qhov tseem ceeb ntawm kev suav lej ntawm qhov sib txawv ntawm cov qib kev qhia tawm ntawm cov nroj tsuag uas tau txhaj tshuaj (Colletotrichum lupini, hom Col-08, tau txais xyoo 1999 los ntawm thaj chaw lupin hauv Wierzenica, Poland) thiab cov nroj tsuag tswj (tsom-inoculated) tau cim saum cov ntsiab lus ntaub ntawv (*P tus nqi < 0.05, **P tus nqi ≤ 0.01, ***P tus nqi ≤ 0.001). Qhov tseem ceeb ntawm kev suav lej ntawm qhov sib txawv ntawm cov qib kev qhia tawm ntawm cov nroj tsuag uas tau txhaj tshuaj (Colletotrichum lupini, hom Col-08, tau txais xyoo 1999 los ntawm thaj chaw lupin hauv Wierzenica, Poland) thiab cov nroj tsuag tswj (tsom-inoculated) tau cim saum cov ntsiab lus ntaub ntawv (*P tus nqi < 0.05, **P tus nqi ≤ 0.01, ***P tus nqi ≤ 0.001). Статистическая значимость различий в уровнях экспрессии между инокулированными (Colletotrichum lupini, штамм 9угнем, Col-08 с поля люпина в Верженице, Польша) и контрольными (ложно инокулированными) растениями отмечхена на дича* PAWG 0,05, **значение P ≤ 0,01, ***значение P ≤ 0,001). Qhov sib txawv ntawm cov qib kev qhia tawm ntawm cov nroj tsuag uas tau txhaj tshuaj (Colletotrichum lupini, hom Col-08, tau txais xyoo 1999 los ntawm thaj teb lupine hauv Wierzhenice, Poland) thiab cov nroj tsuag tswj (sham-inoculated) tau sau tseg saum toj no cov ntsiab lus ntaub ntawv (*P tus nqi < 0.05, **P-tus nqi ≤ 0.01, ***P-tus nqi ≤ 0.001).接种 (Colletotrichum lupini, Col-08 Lub Kaum Ob Hlis, 1999, 1999, Wierzenica ntawm Wierzenica )和对照（模拟接种）植物之间表达水平差异的统计学显着性标记在数据点上异（*P 0.05, **P 值≤ 0.01, ***P 值≤ 0.001).接种 (colletotrichum lupini , color-08 株 , 1999 年 波兰 波兰 wierzenica 的 羽扇获得 )差异的统计学 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, люпина в Верженице, Польша, в 1999 г.) и контрольными (ложно инокулированными) растениями отмечены над значение P < 0,05, ** P-значение ≤ 0,01, ***P-значение ≤ 0,001). Qhov sib txawv ntawm cov qib kev qhia tawm ntawm cov nroj tsuag uas tau txhaj tshuaj (Colletotrichum lupini, hom Col-08, tau los ntawm cov teb lupine hauv Verzhenice, Poland, xyoo 1999) thiab cov nroj tsuag tswj (sham-inoculated) tau sau tseg saum toj no cov ntsiab lus ntaub ntawv (*P tus nqi < 0.05, **P-tus nqi ≤ 0.01, ***P-tus nqi ≤ 0.001).Cov kab NLL uas tau soj ntsuam yog: 83A: 476 (tiv taus, nqa cov allele Lanr1 homozygous), Mandelup (tiv taus nruab nrab, nqa cov allele AnMan homozygous), Boregine (tiv taus, tsis paub keeb kwm caj ces) thiab pej xeem 22660 (tiv taus).
Tus gene TanjilG_05042 ntawm Lanr1 locus tau qhia txog tus qauv qhia tawm txawv ntawm cov profiles tau los ntawm RNA-seq kev tshawb fawb (Daim duab 6e). Kev ua haujlwm tseem ceeb ntawm cov gene no tau pom hauv Mandelup thiab cov pej xeem 22660 (txog li 39.7 thiab 11.7 zaug, feem), ua rau muaj cov qib qhia tawm siab (txog li 1.4 ± 0.14 thiab 7.2 ± 1.3, feem). 83A: 476 kuj tau qhia txog qee qhov kev nce ntxiv ntawm TanjilG_05042 gene (txog li 3.8-npaug), txawm li cas los xij, cov qib qhia tawm sib piv tau (0.044 ± 0.002) yog ntau dua 30-npaug qis dua li cov uas tau pom hauv Mandelup thiab cov pej xeem 22660. kev tshuaj xyuas los ntawm qPCR qhia tau qhov sib txawv tseem ceeb hauv cov qib kev qhia tawm ntawm cov genotypes hauv cov qauv tshuaj tiv thaiv (tswj), ncav cuag qhov sib txawv 58 npaug ntawm cov pej xeem 22660 thiab 83A: 476, nrog rau ntawm cov pej xeem 22660 thiab 22660. Qhov sib txawv ob npaug tau ua tiav ntawm Boregine thiab Mandalup.
Tus gene sib tw ntawm AnMan locus, TanjilG_12861, tau qhib ua teb rau kev txhaj tshuaj hauv 83A: 476 thiab Mandelup, yog qhov nruab nrab hauv cov pej xeem 22660, thiab tau downregulated hauv Boregine (Daim duab 6f). Qhov kev qhia txog TanjilG_12861 gene yog qhov siab tshaj plaws hauv inoculated 83A: 476 (0.14 ± 0.01). Tus 17.4 kDa chav kawm I heat shock protein gene TanjilG_05080 HSP17.4 qhia cov qib kev qhia qis dua hauv txhua hom kab mob thiab lub sijhawm kawm (Daim duab 6g). Tus nqi siab tshaj plaws tau pom ntawm 24 HPI hauv cov pej xeem 22660 (0.14 ± 0.02, qhov nce yim npaug hauv kev teb rau kev txhaj tshuaj).
Kev sib piv ntawm cov qauv qhia txog cov noob caj noob ces (Daim Duab 7) tau qhia txog kev sib raug zoo siab ntawm TanjilG_10657 thiab plaub lwm cov noob caj noob ces: TanjilG_27015 (r = 0.89), TanjilG_05080 (r = 0.85), TanjilG_05042 (r = 0.80), thiab TanjilG_04706 (r = 0.79). Cov txiaj ntsig zoo li no yuav qhia tau tias muaj kev sib koom ua ke ntawm cov noob caj noob ces no thaum lub sijhawm tiv thaiv teb. Cov noob caj noob ces TanjilG_12861 thiab TanjilG_23384 tau qhia txog cov qauv qhia sib txawv nrog cov nqi qis dua Pearson correlation coefficient (los ntawm 0.08 txog 0.43 thiab -0.19 txog 0.28, feem) piv rau lwm cov noob caj noob ces.
Kev sib raug zoo ntawm cov qauv qhia txog kev qhia txog noob caj noob ces tau raug kuaj pom los ntawm kev siv PCR kom paub ntau yam. Cov kab lupine nplooj nqaim hauv qab no tau raug tshuaj xyuas: 83A:476 (tiv taus, nqa cov allele Lanr1 homozygous), Mandelup (tiv taus nruab nrab, nqa cov allele AnMan homozygous), Boregine (tiv taus, tsis paub keeb kwm yav dhau los ntawm noob caj noob ces), thiab Population 22660 (tiv taus). Peb lub sijhawm tau raug suav (6, 12 thiab 24 teev tom qab txhaj tshuaj), suav nrog cov nroj tsuag uas tau txhaj tshuaj (Colletotrichum lupini, hom Col-08, tau los ntawm cov teb lupine hauv Wierzhenice, Poland, xyoo 1999) thiab cov nroj tsuag tswj (sham-inoculated). Lub nplai qhia txog tus nqi ntawm Pearson correlation coefficient.
Raws li cov ntaub ntawv tau txais ntawm 6 horsepower ib nti, WGCNA tau ua tiav ntawm 9981 DEG txheeb xyuas los ntawm kev sib piv cov nroj tsuag inoculated thiab cov nroj tsuag tswj kom tsom mus rau cov lus teb tiv thaiv thaum ntxov (Supplementary Table S12). Nees nkaum ob lub gene modules (clusters) tau pom nrog cov qauv qhia tawm sib raug zoo (zoo lossis tsis zoo) ntawm genotypes thiab cov kev hloov pauv sim. Qhov nruab nrab, cov theem kev qhia tawm ntawm cov noob caj noob ces tau nqis los hauv kev txiav txim 83A: 476 > Mandelup > Boregine > Population 22660 (hauv ob qho kev hloov pauv, txawm li cas los xij, qhov sib txawv no muaj zog dua hauv cov nroj tsuag tswj). Qhov nruab nrab, cov theem kev qhia tawm ntawm cov noob caj noob ces tau nqis los hauv kev txiav txim 83A: 476 > Mandelup > Boregine > Population 22660 (hauv ob qho kev hloov pauv, txawm li cas los xij, qhov sib txawv no muaj zog dua hauv cov nroj tsuag tswj). В среднем уровни экспрессии генов снижались в порядке 83A:476 > Mandelup > Boregine > Population 22660 (в обоих вариантах , о днантах , о дната си была сильнее у контрольных растений). Qhov nruab nrab, cov qib kev qhia tawm ntawm cov noob caj noob ces tau txo qis hauv qhov kev txiav txim 83A: 476 > Mandelup > Boregine > Population 22660 (hauv ob qho kev hloov pauv, txawm li cas los xij, qhov sib txawv no muaj zog dua hauv cov nroj tsuag tswj).平均而言，基因表达水平按83A:476 > Mandelup > Boregine > Population 22660的顺序下降（然而，在两种变体中，这种趋势在对照植物中更强）.平均而言，基因水平按按 83a: 476> mandelup> boregine> pejxeem 22660 的顺序 下降在（，在种丙 ，中更) . . . . . . . . . . . . . . . . . . . . . . . В среднем уровни экспрессии генов снижались в ряду 83A:476 > Mandelup > Boregine > Population 22660 (однако в обоих вариант ах сильнее у контрольных растений). Qhov nruab nrab, cov qib kev qhia tawm ntawm cov noob caj noob ces tau txo qis hauv cov koob 83A: 476 > Mandelup > Boregine > Population 22660 (txawm li cas los xij, hauv ob qho tib si, qhov sib txawv no muaj zog dua hauv cov nroj tsuag tswj).Kev txhaj tshuaj tiv thaiv kab mob ua rau muaj kev nce ntxiv ntawm kev qhia tawm ntawm cov noob caj noob ces, tshwj xeeb tshaj yog nyob rau hauv cov modules 18, 19, 14, 6 thiab 1 (hauv kev txiav txim siab ntawm cov nyhuv), kev tswj hwm tsis zoo (piv txwv li cov modules 9 thiab 20) lossis nrog cov teebmeem nruab nrab (piv txwv li cov modules 11, 22, 8 thiab 13). GO lub sijhawm kev tshuaj xyuas kev nplua nuj (Supplementary Table S13) tau qhia "GO: 0006952 Cov lus teb tiv thaiv" rau cov module inoculated (18) nrog kev ua haujlwm siab tshaj plaws, suav nrog cov noob caj noob ces uas tau tshuaj xyuas los ntawm qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 thiab TanjilG_27015), nrog rau ntau cov modules photosynthesis uas raug txwv tsis pub siv Inoculate (9). Module 18 concentrator (Daim Duab 8) tau txheeb xyuas tias yog TanjilG_26536 gene uas encoded cov PR-10-zoo li LlR18B protein, thiab module 9 concentrator tau txheeb xyuas tias yog TanjilG_28955 gene uas encoded cov photosystem II PsbQ protein. Ib tug neeg sib tw anthracnose resistance gene Lanr1, TanjilG_05042, tau pom nyob rau hauv module 22 (Daim Duab 9) thiab cuam tshuam nrog cov lus "GO:0044260 Cellular macromolecular metabolic processes" thiab "GO:0006355 Transcriptional regulation, DNA templating" nqa TanjilG_01212 hub. lub gene encodes heat stress transcription factor A-4a (HSFA4a).
Kev tshuaj xyuas lub network hnyav ntawm kev sib koom ua ke ntawm cov noob caj noob ces ntawm cov modules nrog cov txheej txheem ntawm cov txheej txheem biological uas tau sawv cev ntau dhau "GO: 0006952 Cov lus teb tiv thaiv". Kev sib txuas tau yooj yim kom pom tseeb plaub lub noob caj noob ces uas tau tshuaj xyuas los ntawm qPCR (TanjilG_04706, TanjilG_23384, TanjilG_10657 thiab TanjilG_27015).
Kev tshuaj xyuas lub network hnyav ntawm kev sib koom ua ke ntawm cov noob caj noob ces ntawm ib lub module nrog cov txheej txheem biological uas muaj ntau dhau los ua "GO: 0006355: Kev tswj hwm kev sau ntawv, DNA templating" thiab nqa tus neeg sib tw anthracnose tiv thaiv gene Lanr1 TanjilG_05042. Ligation tau yooj yim dua los cais cov noob caj noob ces TanjilG_05042 thiab cov noob caj noob ces TanjilG_01212 nruab nrab.
Kev tshuaj xyuas kev tiv thaiv anthracnose uas tau sau los ntawm Australia qhia tau tias feem ntau ntawm cov cultivars uas tso tawm thaum ntxov yog qhov tsis haum; Kalya, Coromup thiab Mandelup tau piav qhia tias muaj kev tiv thaiv nruab nrab, thaum Wonga, Tanjil thiab 83A: 476 tau piav qhia tias muaj kev tiv thaiv siab heev26,27,31. muaj tib lub allele tiv thaiv, xaiv Lanr1, thiab Coromup thiab Mandelup muaj ib lub allele sib txawv, xaiv AnMan10, 26, 39, thaum Kalya dhau mus rau ib lub allele sib txawv. , Lanr2. Kev tshuaj xyuas kev tiv thaiv anthracnose hauv lub teb chaws Yelemees tau ua rau pom cov kab tiv thaiv Bo7212 nrog tus neeg sib tw allele uas tsis yog Lanr1, xaiv LanrBo36.
Peb txoj kev tshawb fawb tau qhia txog qhov tsawg heev (kwv yees li 6%) ntawm Lanr1 allele hauv cov noob caj noob ces uas tau sim. Qhov kev soj ntsuam no yog sib xws nrog cov txiaj ntsig ntawm kev tshuaj ntsuam East European germplasm siv cov cim Anseq3 thiab Anseq4, uas qhia tau tias Lanr1 allele tsuas yog muaj nyob rau hauv ob kab Belarusian xwb. Qhov no qhia tau hais tias Lanr1 allele tseem tsis tau siv dav los ntawm cov kev pab cuam yug me nyuam hauv zos, tsis zoo li hauv Australia, qhov twg nws yog ib qho ntawm cov alleles tseem ceeb rau kev yug me nyuam uas tau pab los ntawm cov cim. Qhov no yuav yog vim qib qis dua ntawm kev tiv thaiv los ntawm Lanr1 allele hauv cov xwm txheej hauv thaj chaw European piv rau daim ntawv tshaj tawm Australian. Tsis tas li ntawd, kev tshawb fawb txog anthracnose hauv thaj chaw muaj nag ntau hauv Australia tau qhia tias cov lus teb tiv thaiv los ntawm Lanr1 allele yuav tsis zoo rau cov huab cua uas nyiam kev loj hlob thiab kev loj hlob sai ntawm cov kab mob pathogen19,42. Qhov tseeb, hauv kev tshawb fawb tam sim no, qee cov tsos mob ntawm anthracnose kuj tau pom hauv cov genotypes nqa Lanr1 allele, qhia tias kev tiv thaiv yuav ploj mus rau hauv cov xwm txheej zoo tshaj plaws rau kev loj hlob ntawm C. lupini. Ntxiv rau, kev txhais lus tsis tseeb ntawm qhov muaj cov cim Anseq3 thiab Anseq4, uas yog kwv yees li 1 cM los ntawm Lanr1 locus, yog ua tau 28, 30, 43.
Peb txoj kev tshawb fawb qhia tau hais tias 83A: 476, nqa Lanr1 allele, teb rau C. lupini inoculation nrog cov ntawv sau loj ntawm qhov kev hloov kho tshiab ntawm thawj lub sijhawm soj ntsuam (6 hpi), thaum nyob hauv Mandelup, nqa AnMan allele, cov lus teb transcriptomic tau pom ntau tom qab. (los ntawm 24 txog 48 hp). Cov kev hloov pauv ntawm lub sijhawm no hauv kev tiv thaiv teb yog cuam tshuam nrog qhov sib txawv ntawm cov tsos mob ntawm tus kab mob, qhia txog qhov tseem ceeb ntawm kev paub txog cov kab mob thaum ntxov rau kev teb zoo rau kev tiv thaiv. Txhawm rau kis tus kab mob rau cov ntaub so ntswg ntawm cov nroj tsuag, cov noob anthrax yuav tsum dhau los ntawm ntau theem kev loj hlob ntawm tus tswv tsev, suav nrog kev tawg paj, kev faib cell, thiab kev tsim ntawm appressorium. Ib qho appendage yog ib qho qauv kis kab mob uas txuas rau ntawm qhov chaw ntawm tus tswv tsev thiab pab txhawb kev nkag mus rau hauv cov ntaub so ntswg ntawm tus tswv tsev. Yog li, cov noob ntawm C. gloeosporioides hauv cov taum pauv tau qhia thawj qhov kev faib ntawm lub nucleus tom qab 75-90 feeb ntawm kev sib xyaw, kev tsim ntawm lub raj noob tom qab 90-120 feeb, thiab kev tswj hwm tom qab 4 teev 45. Txiv nkhaus taw C. gloeosporioides tau qhia tias muaj ntau dua 40% ntawm cov kab mob conidial tom qab 3 teev ntawm kev sib kis thiab kwv yees li 20% ntawm kev tsim cov appresors tom qab 4 teev. Cov noob CAP20 uas cuam tshuam nrog kev sib kis ntawm C. gloeosporioides tau qhia txog kev ua haujlwm transcriptional hauv epiphyte-forming conidia tom qab 3.5 teev kev sib kis hauv avocado surface wax nrog cov protein CAP20 ntau tom qab 4 teev 46 feeb. Ib yam li ntawd, kev ua haujlwm ntawm cov noob melanin biosynthesis hauv C. trifolii tau tshwm sim thaum lub sijhawm sib kis 2 teev ua raws li kev tsim cov appressorium tom qab 1 teev. Kev tshawb fawb ntawm cov ntaub so ntswg nplooj tau qhia tias cov txiv pos nphuab uas tau txhaj tshuaj nrog C. acutatum muaj kev txwv thawj zaug ntawm 8 hpi, thaum cov txiv lws suav uas tau txhaj tshuaj nrog C. coccodes muaj kev txwv thawj zaug ntawm 4 hpi48,49. feem ntau sib xws nrog lub sijhawm ntawm Colletotrichum spp. cov txheej txheem kis kab mob. Cov lus teb tiv thaiv sai rau 83A: 476 qhia txog kev koom tes ntawm cov nroj tsuag tiv thaiv thiab cov noob tiv thaiv kab mob (ETI) hauv kab no, thaum Mandelup cov lus teb qeeb txhawb nqa qhov kev xav ntawm micro-associated molecular pattern-triggered immunity (MTI) 50. Cov lus teb thaum ntxov rau 83A: 476 thiab Mandelup. Qhov sib tshooj ib nrab ntawm cov noob caj noob ces nce lossis nqis hauv kev teb qeeb kuj txhawb nqa lub tswv yim no, vim ETI feem ntau suav hais tias yog kev teb MTI sai thiab txhim kho uas ua rau muaj kev tuag ntawm cov hlwb programmed ntawm qhov chaw kis kab mob, hu ua anaphylactic shock 51,52.
Feem ntau ntawm cov noob caj noob ces uas tau muab piv rau lo lus Gene Ontology GO:0006952 "Defense Response" yog 11 homologues ntawm cov protein fasting message 22 uas ua rau muaj kev ntxhov siab (zoo ib yam li SAM22) thiab xya cov protein latex loj (MLPs). -zoo li cov protein 31, 34, 43 thiab 423 tau qhia txog kev sib xws. Cov noob caj noob ces zoo li SAM22 tau qhia txog kev ua haujlwm tseem ceeb uas kav ntev dua, qhia txog qib kev tiv thaiv anthracnose ntau ntxiv (83A:476 thiab Boregine). Txawm li cas los xij, cov noob caj noob ces zoo li MLP tsuas yog raug txo qis hauv cov kab uas nqa cov allele tiv thaiv tus neeg sib tw (83A:476/Lanr1 ntawm 6 hpi thiab Mandelup/AnMan ntawm 24 hpi). Yuav tsum nco ntsoov tias txhua qhov sib xws zoo li SAM22 tau los ntawm ib pawg noob caj noob ces uas muaj li ntawm 105 kb, thaum cov noob caj noob ces zoo li MLP tau los ntawm thaj chaw sib cais ntawm cov genome. Kev sib koom ua ke ntawm cov noob caj noob ces zoo li SAM22 kuj tau pom nyob rau hauv peb txoj kev tshawb fawb yav dhau los ntawm NLL tiv thaiv Diaporthetoxica inoculation, qhia tias lawv koom nrog cov khoom kab rov tav ntawm kev tiv thaiv teb. Qhov xaus no kuj tau txhawb nqa los ntawm cov ntawv ceeb toom ntawm kev teb zoo ntawm cov noob caj noob ces zoo li SAM22 rau kev raug mob lossis kev kho mob nrog salicylic acid, fungal inducers, lossis hydrogen peroxide.
Cov noob caj noob ces zoo li MLP tau pom tias teb rau ntau yam kev ntxhov siab abiotic thiab biotic, suav nrog cov kab mob bacterial, viral thiab pathogenic fungal kab mob hauv ntau hom nroj tsuag55. Cov lus qhia ntawm kev teb rau qee qhov kev sib cuam tshuam ntawm cov nroj tsuag thiab cov kab mob sib txawv ntawm kev nce ntxiv (piv txwv li, thaum muaj kab mob ntawm paj rwb nrog Verticillium dahliae) mus rau qhov txo qis (piv txwv li, tom qab muaj kab mob ntawm tsob ntoo kua nrog Alternaria spp.)56,57. Kev txo qis tseem ceeb ntawm MLP-zoo li 423 gene tau pom thaum lub sijhawm tiv thaiv avocado rau F. niger kab mob thiab thaum muaj kab mob ntawm tsob ntoo kua Botryosphaeria berengeriana f. cn. piricola thiab Alternaria alternata yog cov kab mob kua58,59. Tsis tas li ntawd, kua calli overexpressing MLP-zoo li 423 gene muaj kev qhia qis dua ntawm cov noob caj noob ces tiv taus thiab muaj kev cuam tshuam ntau dua rau kev kis kab mob fungal59. Tom qab Fusarium oxysporum f, MLP-zoo li 423 gene kuj tau suppressed hauv cov noob caj noob ces tiv taus. cn. Taum kab mob 60.
Lwm cov tswv cuab ntawm tsev neeg PR-10 uas tau txheeb xyuas hauv peb txoj kev tshawb fawb RNA-seq yog cov noob LlR18A thiab LlR18B uas teb rau kev nce siab, nrog rau cov noob nce siab (1 noob) lossis qis dua (3 noob) rau cov protein hloov pauv lipid DIR1. . Tsis tas li ntawd, WGCNA qhia txog LlR18B noob ua lub hauv paus hauv qhov module no, uas yooj yim rau kev txhaj tshuaj tiv thaiv thiab nqa ntau cov noob teb tiv thaiv. Cov noob LlR18A thiab LlR18B tau raug tsim hauv nplooj lupine daj los teb rau cov kab mob pathogenic, nrog rau hauv NLL stems tom qab D. toxica inoculation, thaum cov mov homologue ntawm cov noob no, RSOsPR10, tau raug tsim sai sai los ntawm kev kis tus kab mob fungal uas cuam tshuam nrog txoj kev jasmonic acid signaling pathway53,61,62. Cov noob DIR1 encodes cov protein thauj lipid tsis tshwj xeeb uas xav tau rau qhov pib ntawm kev tiv thaiv kab mob systemic (SAR). Nrog rau kev txhim kho ntawm cov tshuaj tiv thaiv, cov protein DIR1 raug thauj los ntawm qhov chaw ntawm kev kis kab mob los ntawm phloem kom ua rau SAR hauv cov kab mob deb. Qhov nthuav yog, TanjilG_02313 DIR1 gene tau raug tsim los ntawm thawj lub sijhawm hauv kab 84A: 476 thiab Population 22660, tab sis kev tiv thaiv anthracnose tau tsim kho zoo tsuas yog hauv kab 84A: 476. Qhov no yuav qhia txog qee qhov kev ua haujlwm ntawm DIR1 gene hauv NLL, txij li peb qhov sib xws tseem ceeb tau teb rau kev txhaj tshuaj tsuas yog hauv kab 83A: 476 ntawm 6 hpi, thiab cov lus teb no tau taw qhia rau hauv qab.
Hauv peb txoj kev tshawb fawb, cov khoom sib xyaw feem ntau uas sib raug rau cov txheej txheem biological hu ua "GO: 0055114 Redox process" yog cytochrome P450 protein, peroxidase, linoleic acid 9S- / 13S-lipoxygenase, thiab 1-aminocyclopropane-1-carboxylic acid oxidase. Tsis tas li ntawd, peb WGCNA txhais HSFA4a homologue ua lub hub nqa modules xws li Lanr1 resistance gene candidate TanjilG_05042. HSFA4a yog ib feem ntawm redox-dependent kev tswj hwm ntawm nuclear transcription hauv cov nroj tsuag.
Cov protein Cytochrome P450 yog oxidoreductases uas catalyze NADPH thiab / lossis O2-dependent hydroxylation reactions hauv thawj thiab theem nrab metabolism, suav nrog cov metabolism ntawm xenobiotics, nrog rau cov tshuaj hormones, fatty acids, sterols, cell phab ntsa Cheebtsam, biopolymers, thiab biosynthesis ntawm cov tshuaj tiv thaiv 69. Hauv peb txoj kev tshawb fawb, qhov sib txawv ntawm cov nroj tsuag cytochrome P450 ua haujlwm tau txo qis los ntawm -10.6 log2 (fold hloov) mus rau 5.7 vim muaj ntau tus hloov pauv homologues (37) thiab qhov sib txawv ntawm cov qauv teb ntawm cov noob tshwj xeeb, uas cuam tshuam txog kev kho dua tshiab. . Siv tsuas yog RNA-seq cov ntaub ntawv los piav qhia txog kev ua haujlwm ntawm cov noob NLL hauv cov protein loj superfamily yuav yog qhov xav paub ntau heev. Txawm li cas los xij, nws tsim nyog sau tseg tias qee cov noob cytochrome P450 cuam tshuam nrog kev tiv thaiv ntau ntxiv rau cov kab mob pathogenic fungi lossis cov kab mob, suav nrog kev pab txhawb rau kev ua xua 69,70,71.
Chav Kawm III peroxidases yog cov enzymes cog ntau yam uas koom nrog ntau yam txheej txheem metabolic thaum lub sijhawm cog qoob loo loj hlob thiab txhim kho, nrog rau kev teb rau kev ntxhov siab ib puag ncig xws li salinity, av qhuav, lub teeb ci ntau, thiab kev tawm tsam kab mob72. Peroxidases koom nrog kev sib cuam tshuam ntawm ntau hom nroj tsuag nrog Anthracis, suav nrog Stylosanthes humilis thiab C. gloeosporioides, Lens culinaris thiab C. truncatum, Phaseolus vulgaris thiab C. lindemuthianum, Cucumis sativus thiab C. lagenarium73,74,75,76. Cov lus teb yog sai heev, qee zaum txawm tias ntawm 4 HPI, ua ntej cov pwm nkag mus rau hauv cov ntaub so ntswg cog73. Lub noob peroxidase kuj teb rau D. toxica NLL inoculation. Ntxiv rau lawv cov haujlwm ib txwm muaj los tswj kev tawg oxidative lossis tshem tawm kev ntxhov siab oxidative, peroxidases tuaj yeem cuam tshuam kev loj hlob ntawm kab mob los ntawm kev tsim cov teeb meem lub cev raws li kev txhawb nqa phab ntsa cell thaum lub sijhawm lignification, subunit lossis cross-linking ntawm cov tshuaj tshwj xeeb. Qhov kev ua haujlwm no tuaj yeem raug suav hais tias yog silico rau TanjilG_03329 gene encoding ib qho putative lignin-forming anion peroxidase uas tau upregulated ntau heev hauv peb txoj kev tshawb fawb hauv 83A: 476 resistant kab ntawm 6 HPI, tab sis tsis yog hauv lwm hom thiab lub sijhawm uas tsis teb.
9S-/13S-lipoxygenase ntawm linoleic acid yog thawj kauj ruam hauv txoj kev oxidative ntawm lipid biosynthesis78. Cov khoom ntawm txoj kev no muaj ntau yam haujlwm hauv kev tiv thaiv cov nroj tsuag, suav nrog kev txhawb nqa phab ntsa ntawm tes los ntawm kev tsim cov callose thiab pectin deposits, thiab kev tswj hwm kev ntxhov siab oxidative los ntawm kev tsim cov reactive oxygen species79,80,81,82,83. Hauv kev tshawb fawb tam sim no, kev qhia tawm ntawm linoleic acid 9S-/13S-lipoxygenase tau hloov pauv hauv txhua hom kab mob, tab sis hauv cov pej xeem uas raug kev txom nyem 22660, kev nce qib tau kov yeej ntawm ntau lub sijhawm sib txawv, thaum nyob rau hauv cov kab mob nqa Lanr1 tiv taus thiab AnMan allele, nws hais txog kev sib txawv ntawm cov txheej oxylipin hauv kev tiv thaiv anthrax ntawm cov genotypes no.
Tus 1-aminocyclopropane-1-carboxylate oxidase (ACO) homologue tau nce siab heev (9 noob) lossis txo qis (2 noob) thaum txhaj tshuaj nrog lupine. Tsuas yog ob qho kev zam, tag nrho cov lus teb no tau tshwm sim ntawm 6 hp. ntawm 83A: 476. Cov tshuaj tiv thaiv enzymatic uas ACO cov protein ua yog cov kauj ruam txwv tus nqi hauv kev tsim ethylene thiab yog li ntawd tau tswj hwm heev84. Ethylene yog cov tshuaj hormones cog uas ua ntau yam haujlwm hauv kev tswj hwm kev loj hlob ntawm cov nroj tsuag thiab teb rau cov xwm txheej kev ntxhov siab abiotic thiab biotic. Kev ua kom ACO transcription thiab kev ua haujlwm ntawm txoj kev ethylene signaling yog koom nrog kev nce qib ntawm kev tiv thaiv ntawm cov nplej rau hemibiotrophic fungus oryzae oryzae los ntawm kev tswj hwm kev tsim cov tshuaj reactive oxygen thiab phytoalexins. Ib qho txheej txheem kab mob zoo sib xws uas pom muaj nyob nruab nrab ntawm M. oryzae thiab C. lupini88,89, tawm tsam keeb kwm yav dhau los ntawm kev nce siab ntawm ACO homologues hauv kab 83A: 476 uas tau tshaj tawm hauv kev tshawb fawb no, hloov qhov ua tau ntawm kev tiv thaiv rau NLL anthracnose Ethylene ua ib kauj ruam tseem ceeb hauv cov txheej txheem molecular.
Hauv kev tshawb fawb tam sim no, kev txwv ntau ntawm ntau cov noob caj noob ces uas cuam tshuam nrog photosynthesis tau pom ntawm 6 hpi hauv 83A: 476 thiab ntawm 48 hpi hauv Mandeloop thiab 22660 cov pej xeem. Qhov dav thiab kev nce qib ntawm cov kev hloov pauv no yog sib npaug rau qib. Kev tiv thaiv Anthracnose tau pom hauv qhov kev sim no. Tsis ntev los no, kev txwv muaj zog thiab thaum ntxov ntawm cov ntawv sau txog photosynthesis tau tshaj tawm hauv ntau tus qauv ntawm kev sib cuam tshuam ntawm cov nroj tsuag-kab mob, suav nrog cov kab mob pathogenic thiab fungi. Kev maj mam (los ntawm 2 HPI hauv qee qhov kev sib cuam tshuam) thiab kev txwv thoob ntiaj teb ntawm cov noob caj noob ces uas cuam tshuam nrog photosynthesis hauv kev teb rau kev kis kab mob tuaj yeem ua rau cov nroj tsuag tiv thaiv kab mob raws li kev xa tawm ntawm cov hom oxygen reactive thiab lawv kev sib cuam tshuam nrog txoj kev salicylic acid los daws cov kev tsis haum tshuaj 90,94.
Xaus lus, cov txheej txheem tiv thaiv teb uas tau pom zoo rau cov kab mob tiv taus tshaj plaws (83A: 476) suav nrog kev paub sai sai ntawm cov kab mob los ntawm R gene (tej zaum TIR-NBS-LRR TanjilG_05042) thiab kev teb tsis haum salicylic acid thiab ethylene signaling, ua raws li kev tsim kom muaj SAR ntev. kev ua yog txhawb nqa los ntawm DIR-1 protein. Nws yuav tsum tau sau tseg tias lub sijhawm biotrophic rau C. lupini tus kab mob luv heev (kwv yees li 2 hnub), ua raws li necrotic growth95. Kev hloov pauv ntawm cov theem no yuav cuam tshuam nrog necrosis thiab kev qhia tawm ntawm ethylene-inducible proteins uas ua rau muaj kev cuam tshuam hypersensitivity hauv cov nroj tsuag tswv tsev. Yog li ntawd, lub sijhawm rau kev ntes tau zoo ntawm C. lupini ntawm theem biotrophic yog nqaim heev. Kev rov ua dua ntawm cov noob cuam tshuam nrog redox thiab photosynthesis pom hauv 83A: 476 ntawm 6 hpi yog sib xws nrog kev nce qib ntawm fungal hyphae thiab qhia txog kev txhim kho ntawm kev tiv thaiv zoo ntawm theem biotrophic. Cov lus teb transcriptomic ntawm Mandelup thiab 22660 pej xeem yuav qeeb dhau los ntes cov pwm ua ntej hloov mus rau kev loj hlob necrotic, txawm li cas los xij, Mandelup yuav ua tau zoo dua li 22660 pej xeem vim tias kev tswj hwm sai ntawm PR-10 protein txhawb nqa kev tiv thaiv kab rov tav.
ETI, uas yog los ntawm cov noob caj noob ces R, zoo li yog ib qho uas ua rau taum tsis kam kab mob anthracnose. Yog li ntawd, hauv cov qauv legume Medicago truncatula, kev tiv thaiv kab mob anthracnose yog muab los ntawm cov noob caj noob ces RCT1, ib tug tswv cuab ntawm TIR-NBS-LRR97 cog R gene chav kawm. Cov noob caj noob ces no kuj muab kev tiv thaiv kab mob anthracnose dav dav hauv alfalfa thaum hloov mus rau cov nroj tsuag uas yoo mob. Hauv cov taum feem ntau (P. vulgaris), ntau tshaj ob kaum ob cov noob caj noob ces tiv thaiv kab mob anthracnose tau txheeb xyuas txog niaj hnub no. Ib txhia ntawm cov noob caj noob ces no pom muaj nyob rau hauv thaj chaw uas tsis muaj cov noob caj noob ces R, txawm li cas los xij ntau lwm tus nyob ntawm ntug ntawm cov chromosomes uas nqa cov noob caj noob ces NBS-LRR, suav nrog TIR-NBS-LRRs99. Kev tshawb fawb SSR thoob plaws genome kuj tau lees paub qhov kev sib txuas ntawm cov noob caj noob ces NBS-LRR nrog kev tiv thaiv kab mob anthracnose hauv cov taum feem ntau. Cov noob caj noob ces R kuj pom muaj nyob rau hauv thaj chaw genomic uas nqa qhov chaw tiv thaiv kab mob anthracnose tseem ceeb hauv cov lupine dawb 101.
Peb txoj haujlwm qhia tau hais tias kev tiv thaiv tam sim ntawd, ua haujlwm thaum ntxov ntawm kev kis tus kab mob ntawm cov nroj tsuag (qhov zoo tshaj plaws tsis pub dhau 12 hpi), tiv thaiv tau zoo lupine nplooj nqaim los ntawm anthracnose los ntawm cov pwm pathogenic Collelotrichum lupini. Siv cov txheej txheem siab throughput, peb tau ua qauv qhia txog cov qauv qhia sib txawv ntawm cov noob tiv thaiv anthracnose hauv cov nroj tsuag NLL uas tau txais los ntawm cov noob tiv thaiv Lanr1 thiab AnMan. Kev tiv thaiv zoo suav nrog kev tsim cov noob rau cov protein uas koom nrog redox, photosynthesis, thiab pathogenesis hauv ob peb teev tom qab cov nroj tsuag thawj zaug sib cuag nrog cov kab mob. Cov tshuaj tiv thaiv zoo sib xws, tab sis qeeb hauv lub sijhawm, tsis tshua muaj txiaj ntsig zoo hauv kev tiv thaiv cov nroj tsuag ntawm cov kab mob. Kev tiv thaiv anthrax uas tau txais los ntawm cov noob Lanr1 zoo ib yam li cov lus teb sai ntawm cov noob R (kev tiv thaiv effector-triggered), thaum cov noob AnMan feem ntau muab cov lus teb kab rov tav (kev tiv thaiv kab mob uas tshwm sim los ntawm cov qauv molecular cuam tshuam nrog microbe), muab qib nruab nrab ntawm kev ruaj khov.
Cov kab NLL 215 uas siv los tshuaj xyuas cov cim anthracnose muaj 74 hom kab mob sib txawv, 60 hom kab mob tau los ntawm kev sib xyaw lossis kev yug me nyuam, 5 hom kab mob hloov pauv, thiab 76 hom kab mob qus lossis cov kab mob qub. Cov kab mob no los ntawm 17 lub teb chaws, feem ntau yog los ntawm Poland (58), Spain (47), Lub Tebchaws Yelemees (27), Australia (26), Russia (19), Belarus (7), Ltalis (5) thiab lwm hom kab mob. los ntawm 10 lub teb chaws. Cov txheej txheem kuj suav nrog cov kab mob tiv taus kev siv: 83A: 476, Tanjil, Wonga nqa Lanr1 allele, thiab Mandelup nqa AnMan allele. Cov kab mob no tau txais los ntawm European Lupine Genetic Resource Database uas Poznań Plant Breeding Ltd., Wiatrowo, Poland (Supplementary Table S1).
Cov nroj tsuag tau cog rau hauv qab kev tswj hwm (photoperiod 16 teev, kub 25 ° C thaum nruab hnub thiab 18 ° C thaum hmo ntuj). Ob qhov kev rov ua dua ntawm cov kab mob tau raug tshuaj xyuas. DNA tau raug rho tawm ntawm cov nplooj peb lub lis piam siv DNeasy Plant Mini Kit (Qiagen, Hilden, Lub Tebchaws Yelemees) raws li cov txheej txheem. Qhov zoo thiab qhov concentration ntawm DNA raug rho tawm tau raug soj ntsuam los ntawm cov txheej txheem spectrophotometric (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, Tebchaws USA). Lub cim AnManM1 cim lub noob tiv thaiv anthracnose AnMan (los ntawm cv. Mandelup) thiab cov cim Anseq3 thiab Anseq4 flanking lub noob Lanr1 (los ntawm cv. Tanjil) tau raug tshuaj xyuas 11,26,28. Homozygotes rau cov allele tiv thaiv tau raug qhab nia ua "1", raug - ua "0", thiab heterozygotes - ua 0.5.
Raws li cov txiaj ntsig ntawm kev tshuaj xyuas cov cim AnManM1, AnSeq3 thiab AnSeq4 thiab qhov muaj noob rau kev sim zaum kawg, 50 kab NLL tau raug xaiv rau kev tiv thaiv anthracnose phenotyping. Kev tshuaj xyuas tau ua ob zaug hauv lub tsev cog khoom tswj hwm los ntawm lub khoos phis tawj nrog 14 teev photoperiod nrog qhov kub thiab txias ntawm 22 ° C thaum nruab hnub thiab 19 ° C thaum hmo ntuj. Cov noob raug khawb (txiav cov noob txheej ntawm sab nraud ntawm lub embryo nrog rab riam ntse) ua ntej cog kom tiv thaiv cov noob dormancy vim tias cov noob txheej tawv dhau thiab kom ntseeg tau tias muaj kev sib npaug. Cov nroj tsuag tau cog rau hauv cov lauj kaub (11 × 11 × 21 cm) nrog cov av sterile (TS-1 REC 085 Medium Basic, Klasmann-Deilmann Polska, Warsaw, Poland). Kev txhaj tshuaj tau ua tiav nrog Colletotrichum lupini Col-08 hom, cog rau xyoo 1999 los ntawm cov qia ntawm cov nroj tsuag lupine nplooj nqaim uas cog rau hauv ib daim teb hauv Verzhenitsa, Greater Poland (52° 27′ 42″ N 17° 04′ 05″ E). Tau ib thaj chaw. Cov isolates tau cog rau hauv SNA nruab nrab ntawm 20° C. nyob rau hauv lub teeb dub rau 21 hnub kom induce sporulation. Plaub lub lis piam tom qab sowing, thaum cov nroj tsuag tau mus txog theem 4-6 nplooj, kev txhaj tshuaj tau ua tiav los ntawm kev txau nrog cov tshuaj conidia ntawm qhov concentration ntawm 0.5 x 106 conidia ib ml. Tom qab txhaj tshuaj, cov nroj tsuag tau khaws cia rau hauv qhov tsaus ntuj rau 24 teev ntawm qhov av noo ntawm kwv yees li 98% thiab qhov kub ntawm 25°C kom pab txhawb kev tawg ntawm conidia thiab cov txheej txheem kis kab mob. Cov nroj tsuag tau cog rau hauv 14-teev photoperiod ntawm 22°C hnub / 19°C hmo ntuj thiab 70% av noo. Cov qhab nia kab mob tau ua 22 hnub tom qab txhaj tshuaj thiab nws nyob ntawm 0 (tiv thaiv kab mob) mus rau 9 (muaj feem yuav kis tau kab mob heev) nyob ntawm seb puas muaj lossis tsis muaj cov kab mob necrotic ntawm cov qia thiab nplooj. Tsis tas li ntawd, tom qab ntsuas qhov hnyav ntawm cov nroj tsuag tau ntsuas. Kev sib raug zoo ntawm cov cim genotypes thiab cov qauv kab mob tau suav ua qhov sib txuas ntawm ob kab (tsis muaj cov cim heterozygous hauv cov kab rau kev tshuaj xyuas cov qauv tiv thaiv anthracnose).