Transcriptomic and Expression Analysis of Anthracnose Resistance-related Genes from Yam Suyu 8

doi:10.7668/hbnxb.20192826

Abstract

Abstract:

In this study,the transcription level of resistance genes to the infection of Colletotrichum gloeosporioides were explored at the molecular level and provided candidate genes for further study of disease resistance mechanism.Suyu 8 is a new yam variety highly resistant to anthracnose,which was bred by tissue culture mutagenesis of the highly susceptible strain 024.In order to find out the anthracnose resistance genes,Suyu 8 and strain 024 leaves were inoculated for different time by Colletotrichum gloeosporioides strain 4-2,the differentially expressed genes were analyzed by transcriptome sequencing.Concurrently,the uninoculation leaves of Suyu 8 were used as control.The number of differentially expressed genes,inoculation for 24,48,72,96 h,were 197,132,187 and 313,respectively.After removing the common differentially expressed genes at each time point,we obtained 711 differentially expressed genes.Go enrichment showed that the differential genes were mainly related to response to biological stimulation,defense response,cell wall metabolism and oxidation-reduction process.KEGG enrichment analysis revealed that many metabolic pathways related to plant disease resistance were changed.The expression of multiple genes varied which defense-related plant hormone signal transduction pathways,such as auxin,jasmonic acid,and ethylene.Among them,five early auxin-responsive genes,the key genes of jasmonic acid and abscisic acid biosynthesis were up-regulated,while ERF036 was down-regulated,which might negatively regulate the infection of Colletotrichum gloeosporioides.Several cytochrome P450 genes,ubiquitin ligases involved secondary metabolite modification and phytosterol synthetases,defensins and lectins were up-regulated.WRKY,MYB and TIFY transcription factors positively or negatively regulate the expression of disease resistance genes.Under the regulation of transcription factors,PR protein,NBS-LRR disease resistance genes,receptor kinases were highly expressed,the expressions of CAT and SOD genes of antioxidant protective enzyme systems were activated by reactive oxygen species.In addition,the enzymes related to starch and sucrose synthesis were up-regulated,and enzymes related to starch degradation were down regulated.The expression trends of LAX4,IAA4,IAA21 were consistent with that of transcriptome sequencing.The relative expression of LAX4,IAA4 and IAA21 in resistant varieties were significantly higher than susceptible varieties,suggesting that auxin signaling pathway is beneficial to the immune response to anthracnose for yam.

Key words: Yam, Suyu 8, Anthracnose, Transcriptome, Differentially expressed genes

HAN Xiaoyong, JIANG Lu, YIN Jianmei, JIN Lin, ZHANG Peitong. Transcriptomic and Expression Analysis of Anthracnose Resistance-related Genes from Yam Suyu 8[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 211-222. doi: 10.7668/hbnxb.20192826.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20192826

http://www.hbnxb.net/EN/Y2022/V37/I2/211

Figures/Tables 12

Tab.1 The genes and primers used for Q-PCR

基因 Gene	正向引物(5'—3') Forward primer(5'—3')	反向引物(5'—3') Reverse primer (5'—3')	基因功能描述 Description of gene function
LAX4	CAATCTGGTTCTTCGCCATC	ACGAAGATCCATGCCACCAC	生长素转运类蛋白4
IAA4	AGCAAGCCTCTGATTGGCCAC	CATCTCCAACCATCATCCAATC	生长素响应蛋白4
IAA21	GGATGGAGCTCCTTATCTTAG	CACCGATAGTAAAGCAGCTGA	生长素响应蛋白17
Actin1	CTCATTGATCGGCATGGAAGC	GGGGAACATAGTTGAACCACCAC	肌动蛋白

Tab.2 The statistics of transcriptome sequencing data

样本 Sample	高质量序列 read 数/条 Clean reads	高质量序列碱基数/bp Clean data	GC含量/% GC content	质量值≥20 碱基百分比/% Q20	质量值≥30 碱基百分比/% Q30
R0h	20 504 603	6 151 380 900	45.26	96.30	90.56
S24h	20 225 071	6 067 521 400	45.35	96.18	90.34
R24h	20 160 088	6 048 026 300	45.30	96.27	90.56
S48h	18 874 399	5 662 319 600	45.67	96.13	90.25
R48h	19 875 790	5 962 737 000	45.31	96.30	90.57
S72h	18 538 496	5 561 548 900	45.05	96.24	90.46
R72h	20 022 778	6 006 833 400	44.98	96.32	90.62
S96h	18 567 803	5 570 340 900	45.40	96.16	90.33
R96h	19 518 804	5 855 641 100	44.92	96.26	90.49

Fig.1 The statistics of differentially expressed genes between samples

Fig.2 Venn map of differentially expressed genes
A—D.Venn map of differentially expressed genes after inoculation 24,48,72,96 h, respectively;E.Venn map of common differentially expressed genes after inoculation 24,48,72,96 h.

Fig.3 GO classification of the differentially expressed genes
BP.Biological process:BP1.Cellular process;BP2.Metabolic process;BP3.Single-organism process;BP4.Response to stimulus;BP5.Biological regulation;BP6.Localization;BP7.Cellular component organization or biogenesis;BP8.Developmental process;BP9.Signaling;BP10.Multicellular organismal process;BP11.Reproduction;BP12.Reproductive process;BP13.Multi-organism process;BP14.Immune system process;BP15.Growth;BP16.Detoxification;BP17.Rhythmic process;BP18.Locomotion;BP19.Cell killing;BP20.Biological adhesion.CC.Cellular component:CC1.Cell;CC2.Cell part;CC3.Organelle;CC4.Membrane;CC5.Organelle part;CC6.Membrane part;CC7.Macromolecular complex;CC8.Extracellular region;CC9.Membrane-enclosed lumen;CC10.Cell junction;CC11.Supramolecular complex;CC12.Extracellular region part;CC13.Nucleoid;CC14.Other organism;CC15.Other organism part.MF.Molecular function:MF1.Binding;MF2.Catalytic activity;MF3.Transporter activity;MF4.Structural molecule activity;MF5.Signal transducer activity;MF6.Nucleic acid binding transcription factor activity;MF7.Molecular transducer activity;MF8.Molecular function regulator;MF9.Electron carrier activity;MF10.Antioxidant activity;MF11.Nutrient reservoir activity;MF12.Transcription factor activity, protein binding;MF13.Metallochaperone activity;MF14.Translation regulator activity;MF15.Protein tag.

Fig.4 GO enrichment circular graph of the differentially expressed genes

Fig.5 Top 20 of significant enrichment terms

Fig.6 KEGG enrichment classification of the differentially expressed genes

Tab.3 KEGG pathways significantly enriched of differentially expressed genes

Ko号 Ko ID	代谢途径 Pathway	差异基因数量/个 DEG number	P值 P-value
map00040	戊糖和葡萄糖醛酸转化	5	0.000 105 447
map00010	糖酵解/糖异生	6	0.001 331 203
map00100	类固醇生物合成	3	0.002 675 332
map00960	托烷,哌啶和吡啶生物碱合成	3	0.003 124 671
map04075	植物激素信号转导	5	0.004 122 894
map00052	半乳糖代谢	3	0.007 001 598
map00500	淀粉与蔗糖代谢	4	0.017 638 130
map00030	磷酸戊糖途径	3	0.020 066 528
map00561	甘油酯代谢	3	0.034 833 484
map00780	生物素代谢	2	0.043 858 617

Fig.7 Heatmap of the differentially expressed genes
A.Plant-phytopathogen interaction;B.Starch and sucrose metabolism;C.Hormones;D.Transcription factors;E.Secondary metabolism.

Fig.8 Q-PCR verification of differentially expressed genes

Fig.9 Q-PCR analysis of differentially expressed genes
024-S, 024-R, JXD-S, JXD-R, Su8-R, Su6-R, HH-R show strain 024 susceptible leaf and healthy leaf, Jiangxi yam susceptible leaf and healthy leaf, Suyu 8, Suyu 6 and Honghe yam.

References 51

[1]	Onyeka T J,Petro D,Etienne S,Jacqua G,Ano G.Optimizing controlled environment assessment of levels of resistance to anthracnose disease to yam using tissue culture-derived whole plants[J].Journal of Phytopathology,2006,154(5):286-292.doi:10.1111/j.1439-0434.2006.01095.x.
[2]	Lebot V,Abraham K,Kaoh J,Rogers C,Molisalé T.Development of anthracnose resistant hybrids of the greater yam(Dioscorea alata L.)and interspecific hybrids with D.Nummularia Lam[J].Genetic Resources and Crop Evolution,2019,66(4):871-883.doi:10.1007/s10722-019-00756-y.
[3]	Ntui V O,Uyoh E A,Ita E E,Markson A A A,Tripathi J N,Okon N I,Akpan M O,Phillip J O,Brisibe E A,Ene-Obong E O E,Tripathi L.Strategies to combat the problem of yam anthracnose disease:Status and prospects[J].Molecular Plant Pathology,2021,22(10):1302-1314.doi:10.1111/mpp.13107.
[4]	Narina S S,Buyyarapu R,Kottapalli K R,Sartie A M,Ali M I,Robert A,Hodeba M J D,Sayre B L,Scheffler B E.Generation and analysis of expressed sequence tags(ESTs)for marker development in yam(Dioscorea alata L.)[J].BMC Genomics,2011,12:100.doi:10.1186/1471-2164-12-100.
[5]	Darkwa K,Olasanmi B,Asiedu R,Asfaw A.Review of empirical and emerging breeding methods and tools for yam(Dioscorea spp.)improvement:status and prospects[J].Plant Breeding,2020,139(3):474-497.doi:10.1111/pbr.12783.
[6]	Upadhyaya H D, Wang Y H,Sharma R, Sharma S.Identification of genetic markers linked to anthracnose resistance in sorghum using association analysis[J].Theoretical and Applied Genetics,2013,126(6):1649-1657.doi:10.1007/s00122-013-2081-1.
[7]	Yang S,Gao M,Xu C,Gao J,Deshpande S,Lin S,Roe B A,Zhu H.Alfalfa benefits from Medicago truncatula:The RCT1 gene from M.truncatula confers broad-spectrum resistance to anthracnose in alfalfa[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(34):12164-12169.doi:10.1073/pnas.0802518105.
[8]	Li L,Zhu F Y,Liu H J,Chu A,Lo C.Isolation and expression analysis of defense-related genes in sorghum-Colletotrichum sublineolum interaction[J]. Physiological and Molecular Plant Pathology,2013,84:123-130.doi:10.1016/j.pmpp.2013.08.005.
[9]	张庆雨,刘芳春,段可,王飞,王延秀,高清华.水杨酸对草莓炭疽病响应基因FaNBS20表达的影响[J].园艺学报,2014,41(1):53-62.doi:10.16420/j.issn.0513-353X.2014.01.002.
	Zhang Q Y,Liu F C,Duan K,Wang F,Wang Y X,Gao Q H.Effects of salicylic acid on the expression of FaNBS20 gene responsive to Colletotrichum gloeosporioides infection in Fragaria×ananassa[J].Acta Horticulturae Sinica,2014,41(1):53-62.
[10]	Etxeberria E,Gonzalez P,Achor D,Albrigo G.Anatomical distribution of abnormally high levels of starch in HLB-affected Valencia orange trees[J].Physiological and Molecular Plant Pathology,2009,74(1):76-83.doi:10.1016/j.pmpp.2009.09.004.
[11]	Koh E J,Zhou L J,Williams D S,Park J,Ding N Y,Duan Y P,Kang B H.Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in Citrus leaves infected with Candidatus liberibacter asiaticus[J].Protoplasma,2012,249(3):687-697.doi:10.1007/s00709-011-0312-3.
[12]	Kavalli I H,Slattery C J,Ito H,Okita T W.The conversion of carbon and nitrogen into starch and storage proteins in developing storage organs:an overview[J].Functional Plant Biology,2000,27(6):561-570.doi:10.1071/PP99176.
[13]	Darvill A G,Albersheim P.Phytoalexins and their elicitors-A defense against microbial infection in plants[J].Annual Review of Plant Physiology,1984,35(1):243-275.doi:10.1146/annurev.pp.35.060184.001331.
[14]	贾显禄,王振中,王平.水稻与稻瘟病菌非亲和性互作中重要防御酶活性变化规律研究[J].植物病理学报,2002,32(3):206-213.doi:10.3321/j.issn:0412-0914.2002.03.003.
	Jia X L,Wang Z Z,Wang P.Dynamics of important defense enzyme activity of incompatible interactions between rice and Magnaporthe grisea[J].Acta Phytopathologica Sinica,2002,32(3):206-213.
[15]	Huang H L,Ullah F,Zhou D X,Yi M,Zhao Y.Mechanisms of ROS regulation of plant development and stress responses[J].Frontiers in Plant Science,2019,10:800.doi:10.3389/fpls.2019.00800.
[16]	Hasanuzzaman M,Bhuyan M H M B,Anee T I,Parvin K,Nahar K,Mahmud J A,Fujita M.Regulation of ascorbate-glutathione pathway in mitigating oxidative damage in plants under abiotic stress[J].Antioxidants,2019,8(9):384.doi:10.3390/antiox8090384.
[17]	Torres M A.ROS in biotic interactions[J].Physiologia Plantarum,2010,138(4):414-429.doi:10.1111/j.1399-3054.2009.01326.x.
[18]	Li X,Cong F D,Zhang S L,Zhang D J,Wang X X.A review on recognition of various peroxidases[J].Bioorganic Chemistry,2020,5(1):10-14.doi:10.11648/j.ijbc.20200501.13.
[19]	Tijet N,Helvig C,Pinot F,Le Bouquin R,Lesot A,Durst F,Salaün J P,Benveniste I.Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450(CYP94A1)involved in cutin monomers synthesis[J].The Biochemical Journal,1998,332(Pt 2):583-589.doi:10.1042/bj3320583.
[20]	Yoshida Y,Aoyama Y,Noshiro M,Gotoh O.Sterol 14-demethylase P450(CYP51)provides a breakthrough for the discussion on the evolution of cytochrome P450 gene superfamily[J].Biochemical and Biophysical Research Communications,2000,273(3):799-804.doi:10.1006/bbrc.2000.3030.
[21]	Schaller H.The role of sterols in plant growth and development[J].Progress in Lipid Research,2003,42(3):163-175.doi:10.1016/s0163-7827(02)00047-4.
[22]	Ma M,Wang Q,Li Z J,Cheng H H,Li Z J,Liu X L,Song W N,Appels R,Zhao H X.TaCYP78A3,a gene encoding cytochrome P450 CYP78A3 protein in wheat(Triticum aestivum L.),affects seed size[J].The Plant Journal,2015,83(2):312-325.doi:10.1111/tpj.12896.
[23]	Xu M H,Tang Z S,Tan Y L,Tian Y C,Li C Y,Zhang S H,Chen Z H,Tian W Z.A study on introduction of chitinase gene and beta-1,3-glucanase gene into restorer line of Dian-type hybrid rice(Oryza sativa L.)and enhanced resistance to blast(Magnaporthe grisea)[J].Yi Chuan Xue Bao,2003,30(4):330-334.
[24]	Ang A S W,Cheung R C F,Dan X L,Chan Y S,Pan W L,Ng T B.Purification and characterization of a glucosamine-binding antifungal lectin from Phaseolus vulgaris cv.Chinese pinto beans with antiproliferative activity towards nasopharyngeal carcinoma cells[J].Applied Biochemistry and Biotechnology,2014,172(2):672-686.doi:10.1007/s12010-013-0542-2.
[25]	Shirsekar G,Dai L Y,Hu Y J,Wang X J,Zeng L R,Wang G L.Role of ubiquitination in plant innate immunity and pathogen virulence[J].Journal of Plant Biology,2010,53(1):10-18.doi:10.1007/s12374-009-9087-x.
[26]	E Z,Zhang Y P,Li T T,Wang L,Zhao H M.Characterization of the ubiquitin-conjugating enzyme gene family in rice and evaluation of expression profiles under abiotic stresses and hormone treatments[J].PLoS One,2015,10(4):e0122621.doi:10.1371/journal.pone.0122621.
[27]	Li W,Zhong S H,Li G J,Li Q,Mao B Z,Deng Y W,Zhang H J,Zeng L J,Song F M,He Z H.Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence[J].Cell Research,2011,21(5):835-848.doi:10.1038/cr.2011.4.
[28]	Pandey S P,Somssich I E.The role of WRKY transcription factors in plant immunity[J].Plant Physiology,2009,150(4):1648-1655.doi:10.1104/pp.109.138990.
[29]	Xing D H,Lai Z B,Zheng Z Y,Vinod K M,Fan B F,Chen Z X.Stress-and pathogen-induced Arabidopsis WRKY48 is a transcriptional activator that represses plant basal defense[J].Molecular Plant,2008,1(3):459-470.doi:10.1093/mp/ssn020.
[30]	明聪. 棉花抗枯萎病相关基因GhWRKY48的克隆及功能初步分析[D].石河子:石河子大学,2020.doi:10.27332/d.cnki.gshzu.2020.000633.
	Ming C.Cloning and functional analysis of cotton blight resistance-related gene GhWRKY48[D].Shihezi:Shihezi University,2020.
[31]	刘建芬. GhWRKY48与GhWRKY33基因调控棉花对大丽轮枝菌的抗性反应[D].兰州:甘肃农业大学,2019.
	Liu J F.GhWRKY48 and GhWRKY33 genes regulate cotton resistance to Verticillium dahliae[D].Lanzhou:Gansu Agricultural University,2019.
[32]	Chen L G,Zhang L P,Xiang S Y,Chen Y L,Zhang H Y,Yu D Q.The transcription factor WRKY75 positively regulates jasmonate-mediated plant defense to necrotrophic fungal pathogens[J].Journal of Experimental Botany,2021,72(4):1473-1489.doi:10.1093/jxb/eraa529.
[33]	Cheng H Q,Han L B,Yang C L,Wu X M,Zhong N Q,Wu J H,Wang F X,Wang H Y,Xia G X.The cotton MYB108 forms a positive feedback regulation loop with CML11 and participates in the defense response against Verticillium dahliae infection[J].Journal of Experimental Botany,2016,67(6):1935-1950.doi:10.1093/jxb/erw016.
[34]	Yamada S,Kano A,Tamaoki D,Miyamoto A,Shishido H,Miyoshi S,Taniguchi S,Akimitsu K,Gomi K.Involvement of OsJAZ8 in jasmonate-induced resistance to bacterial blight in rice[J].Plant and Cell Physiology,2012,53(12):2060-2072.doi:10.1093/pcp/pcs145.
[35]	Davletova S,Schlauch K,Coutu J,Mittler R.The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis[J].Plant Physiology,2005,139(2):847-856.doi:10.1104/pp.105.068254.
[36]	Huang X Y,Chao D Y,Gao J P,Zhu M Z,Shi M,Lin H X.A previously unknown zinc finger protein,DST,regulates drought and salt tolerance in rice via stomatal aperture control[J].Genes & Development,2009,23(15):1805-1817.doi:10.1101/gad.1812409.
[37]	Gong S H,Ding Y F,Hu S S,Ding L H,Chen Z X,Zhu C.The role of HD-Zip class I transcription factors in plant response to abiotic stresses[J].Physiologia Plantarum,2019,167(4):516-525.doi:10.1111/ppl.12965.
[38]	Hori Y,Kurotani K I,Toda Y,Hattori T,Takeda S.Overexpression of the JAZ factors with mutated jas domains causes pleiotropic defects in rice spikelet development[J].Plant Signaling & Behavior,2014,9(10):e970414.doi:10.4161/15592316.2014.970414.
[39]	Wang S,Wei X L,Cheng L J,Tong Z K.Identification of a C2H2-type zinc finger gene family from Eucalyptus grandis and its response to various abiotic stresses[J].Biologia Plantarum,2014,58(2):385-390.doi:10.1007/s10535-014-0399-4.
[40]	Wang X F,Ma L,Zhang H L,Zhang Y,Xue M,Ren M Y,Tang K G,Guo H Q,Wang M Y.Constitutive expression of two A-5 subgroup DREB transcription factors from desert shrub Ammopiptanthus mongolicus confers different stress tolerances in transgenic Arabidopsis[J].Plant Cell,Tissue and Organ Culture (PCTOC),2021,147(2):351-363.doi:10.1007/s11240-021-02128-w.
[41]	Tiwari S B, Hagen G, Guilfoyle T.The roles of auxin response factor domains in auxin-responsive transcription[J].The Plant cell,2003,15(2):533-543.doi:10.1105/tpc.008417.
[42]	Fu J,Liu H B,Li Y,Yu H H,Li X H,Xiao J H,Wang S P.Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice[J].Plant Physiology,2011,155(1):589-602.doi:10.1104/pp.110.163774.
[43]	Wang D,Pajerowska-Mukhtar K,Culler A H,Dong X N.Salicylic acid inhibits pathogen growth in plants through repression of the auxin signaling pathway[J].Current Biology,2007,17(20):1784-1790.doi:10.1016/j.cub.2007.09.025.
[44]	Halim V A,Altmann S,Ellinger D,Eschen-Lippold L,Miersch O,Scheel D,Rosahl S.PAMP-induced defense responses in potato require both salicylic acid and jasmonic acid[J].The Plant Journal,2009,57(2):230-242.doi:10.1111/j.1365-313X.2008.03688.x.
[45]	Kusajima M,Yasuda M,Kawashima A,Nojiri H,Yamane H,Nakajima M,Akutsu K,Nakashita H.Suppressive effect of abscisic acid on systemic acquired resistance in tobacco plants[J].Journal of General Plant Pathology,2010,76(2):161-167.doi:10.1007/s10327-010-0218-5.
[46]	Ton J,Mauch-Mani B.β-amino-butyric acid-induced resistance against necrotrophic pathogens is based on ABA-dependent priming for callose[J].The Plant Journal,2004,38(1):119-130.doi:10.1111/j.1365-313X.2004.02028.x.
[47]	Zhang Q Y,Gao M,Wu L W,Wang Y D,Chen Y C.Divergent expression patterns in two Vernicia species revealed the potential role of the Hub gene VmAP2/ERF036 in resistance to Fusarium oxysporum in Vernicia montana[J].Genes,2016,7(12):109.doi:10.3390/genes7120109.
[48]	Balaji V,Smart C D.Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp.michiganensis in transgenic tomato(Solanum lycopersicum)[J].Transgenic Research,2012,21(1):23-37.doi:10.1007/s11248-011-9506-x.
[49]	Mao Z C,Zheng J Y,Wang Y S,Chen G H,Yang Y H,Feng D X,Xie B Y.The new CaSn gene belonging to the snakin family induces resistance against root-knot nematode infection in pepper[J].Phytoparasitica,2011,39(2):151-164.doi:10.1007/s12600-011-0149-5.
[50]	Bindschedler L V,Whitelegge J P,Millar D J,Bolwell G P.A two component chitin-binding protein from French bean-association of a proline-rich protein with a cysteine-rich polypeptide[J].FEBS Letters,2006, 580(6):1541-1546.doi:10.1016/j.febslet.2006.01.079.
[51]	Alonso-Ramírez A,Rodríguez D,Reyes D,Jiménez J A,Nicolás G,López-Climent M,Gómez-Cadenas A,Nicolás C.Evidence for a role of gibberellins in salicylic acid-modulated early plant responses to abiotic stress in Arabidopsis seeds[J].Plant Physiology,2009,150(3):1335-1344.doi:10.1104/pp.109.139352.

Please choose a citation manager

Content to export