Identification of proteins associated with Fusarium crown rot resistance in wheat using label-free quantification analysis

doi:10.1016/S2095-3119(20)63573-0

摘要/Abstract

摘要：

小麦茎基腐病是由镰刀菌属真菌引起的一种世界性土传病害，近年来已严重威胁到我国小麦的安全生产。目前，对该病害的研究主要集中在抗源筛选、抗病位点发掘、接种前后转录组分析等层面。本研究首次采用非标定量蛋白质组学方法，分析比较了我国小麦中抗品种和高感品种在接种前后24、48和72小时后的蛋白质组变化。试验共检测到9234个蛋白质，发掘到783个在接种后不同时间点差异表达的蛋白。对这些差异蛋白的富集分析表明，茎基腐病早期侵染对几丁质代谢、氨基糖代谢、乙醛酸和二元酸代谢，丙氨酸、天冬氨酸和谷氨酸代谢，MAPK信号通路，苯丙素生物合成等重要生物学功能和代谢通路具有显著影响。进一步分析发现共有33种与防御、细胞壁形成和光合作用等功能相关的蛋白质在抗、感品种间接种后多个时间点的表达呈现不同趋势。例如，中抗品种中几丁质酶在接菌后三个时间点均显著上调，而感病品种中接种前后则无显著变化。该酶参与降解真菌细胞壁的重要成分几丁质，从而限制真菌的生长。其它蛋白如糖基转移酶、过氧化物酶等也在中抗品种中特异上调表达。前人报道中，这些蛋白参与了其他重要小麦病害如赤霉病、白粉病等侵染后的应答反应。在感病品种中，小檗碱桥酶等特异下调表达。qRT-PCR试验表明，编码几丁质酶、糖基转移酶、过氧化物酶等8个抗病相关蛋白的基因在抗、感材料间接种前后表达趋势与蛋白质组学结果基本一致。本研究为进一步解析小麦抗茎基腐病分子机制奠定了有力基础，为候选基因筛选提供了重要参考。

Abstract:

Fusarium crown rot (FCR), typically caused by Fusarium pseudograminearum, is a severe soil-borne disease that, in recent years, has become an emerging threat to Chinese wheat crops. For the first time in this study, we investigated and compared the proteomic characteristics of two Chinese wheat varieties (04 Zhong 36 and Xinmai 26) at 24, 48, and 72 h post-inoculation using label-free quantitative proteomic analysis. A total of 9 234 proteins were successfully quantified, of which 783 were differentially expressed after inoculation. These proteins were mainly involved in metabolic, single-organism, and cellular processes. Thirty-three proteins associated with defense, cell wall formation, photosynthesis, etc., showed consistently different expression between the two genotypes at multiple time points. In particular, chitinase, which degrades chitin in the fungal cell wall and limits fungal growth, was exclusively and consistently upregulated in 04 Zhong 36 across the three time points. Other proteins such as flavonoid O-methyltransferase, glycosyltransferase, and peroxidase were only upregulated in 04 Zhong 36, and proteins, including the berberine bridge enzyme and rubisco large subunit-binding protein, were specifically downregulated in Xinmai 26. The expression of transcripts encoding eight selected proteins through qRT-PCR analysis supported the proteomic profiles. Overall, the results of this study allow us to understand FCR resistance in wheat at the protein level. Some proteins and their corresponding genes may be useful resources for the genetic improvement of FCR resistance in wheat.

Key words: wheat , Fusarium crown rot , proteomic , differential expression , multiple time points

. [J]. Journal of Integrative Agriculture, 2021, 20(12): 3209-3221.

JIN Jing-jing, DUAN Shuo-nan, QI Yong-zhi, ZHEN Wen-chao, MA Jun. Identification of proteins associated with Fusarium crown rot resistance in wheat using label-free quantification analysis [J]. Journal of Integrative Agriculture, 2021, 20(12): 3209-3221.

参考文献

Benedetti M, Verrascina I, Pontiggia D, Locci F, Mattei B, De Lorenzo G, Cervone F. 2018. Four Arabidopsis berberine bridge enzyme-like proteins are specific oxidases that inactivate the elicitor-active oligogalacturonides. The Plant Journal (for Cell and Molecular Biology), 94, 260–273.
Berger S, Papadopoulos M, Schreiber U, Kaiser W, Roitsch T. 2004. Complex regulation of gene expression, photosynthesis and sugar levels by pathogen infection in tomato. Physiologia Plantarum, 122, 419–428.
Bernardo L, Prinsi B, Negri A S, Cattivelli L, Espen L, Valè G. 2012. Proteomic characterization of the Rph15 barley resistance gene-mediated defence responses to leaf rust. BMC Genomics, 13, 642.
Bolton M D. 2009. Primary metabolism and plant defense - fuel for the fire. Molecular Plant–Microbe Interaction, 22, 487–497.
Bovill W D, Horne M, Herde D, Davis M, Wildermuth G B, Sutherland M W. 2010. Pyramiding QTL increases seedling resistance to crown rot (Fusarium pseudograminearum) of wheat (Triticum aestivum). Theoretical and Applied Genetics, 121, 127–136.
Bovill W D, Ma W, Ritter K, Collard B C Y, Davis M, Wildermuth G B, Sutherland M W. 2006. Identification of novel QTL for resistance to crown rot in the doubled haploid wheat population ‘W21MMT70’×‘Mendos’. Plant Breeding, 125, 538–543.
Burgess L W, Backhouse D, Summerell B A, Swan L J. 2001. Crown rot of wheat. In: Fusarium: Paul E Nelson Memorial Symposium. The American Phytopathological Society, St Paul, MN. pp. 271–294.
Cheng Q, Li N, Dong L, Zhang D, FanS, Jiang L, Xin W, Xu P, Zhang S. 2015. Overexpression of soybean isoflavone reductase (GmIFR) enhances resistance to Phytophthora sojae in soybean. Frontiers in Plant Science, 6, 1024.
Chou H M, Bundock N, Rolfe S A, Scholes J D. 2000. Infection of Arabidopsis thaliana leaves with Albugo candida (white blister rust) causes a reprogramming of host metabolism. Molecular Plant Pathology, 1, 99–113.
Collard B C Y, Grams R A, Bovill C D, Percy C D, Jolley R, Lehmensiek A, Wildermuth G, Sutherland M W. 2005. Development of molecular markers for crown rot resistance in wheat: Mapping of QTL for seedling resistance in a ‘2-49’×‘Janz’ population. Plant Breeding, 124, 532–537.
Collard B C Y, Jolley R, Bovill W D, Grams R A, Wildermuth G B, Sutherland M W. 2006. Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line ‘2-49’. Australian Journal of Agricultural Research, 57, 967–973.
Daniel B, Pavkov-Keller T, Steiner B, Dordic A, Gutmann A, Nidetzky B, Sensen C W, van der Graaff E, Wallner S, Gruber K, Macheroux P. 2015. Oxidation of monolignols by members of the berberine bridge enzyme family suggests a role in plant cell wall metabolism. Journal of Biological Chemistry, 290, 18770–18781.
Desmond O J, Edgar C I, Manners J M, Maclean D J, Schenk P M, Kazan K. 2005. Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum. Physiological and Molecular Plant Pathology, 67, 171–179.
Desmond O J, Manners J M, Schenk P M, Maclean D J, Kazan K. 2008. Gene expression analysis of the wheat response to infection by Fusarium pseudograminearum. Physiological and Molecular Plant Pathology, 73, 40–47.
Ding L, Li M, Li P, Cao J. 2017. Comparative proteomics analysis of young spikes of wheat in response to Fusarium graminearum infection. Acta Physiologiae Plantarum, 39, 271.
Doehlemann G, Wahl R, Horst R J, Voll L M, Usadel B, Poree F, Stitt M, Pons-Kühnemann J, Sonnewald U, Kahmann R, Kämper J. 2008. Reprogramming a maize plant: Transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis. The Plant Journal: for Cell and Molecular Biology, 56, 181–195.
Egelund J, Skjøt M, Geshi N, Ulvskov P, Petersen B L. 2004. A complementary bioinformatics approach to identify potential plant cell wall glycosyltransferase-encoding genes. Plant Physiology, 136, 2609–2620.
Gunnaiah R, Kushalappa A C, Duggavathi R, Fox S, Somers D J. 2012. Integrated metabolo-proteomic approach to decipher the mechanisms by which wheat QTL (Fhb1) contributes to resistance against Fusarium graminearum. PLoS ONE, 7, e40695.
Guo P, Baum M, Grando S, Ceccarelli S, Bai G, Li R, von Korff M, Varshney R K, Graner A, Valkoun J. 2009. Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage. Journal of Experimental Botany, 60, 3531–3544.
Horst R J, Engelsdorf T, Sonnewald U, Voll L M. 2008. Infection of maize leaves with Ustilago maydis prevents establishment of C4 photosynthesis. Journal of Plant Physiology, 165, 19–28.
Ibrahim R K, Bruneau A, Bantignies B. 1998. Plant O-methyltransferases: Molecular analysis, common signature and classification. Plant Molecular Biology, 36, 1–10.
Jin J J, Duan S N, Qi Y Z, Yan S H, Li W, Li B Y, Xie C J, Zhen W C, Ma J. 2020a. Identification of a novel genomic region associated with resistance to Fusarium crown rot in wheat. Theoretical and Applied Genetics, 133, 2063–2073.
Jin J J, Qi Y Z, Wang L, Wang F F, Yan C M, Li B Y, Xie C J, Zhen W C, Ma J. 2020b. Evaluation of Chinese wheat germplasm resources for crown rot resistance. Journal of Plant Genetic Resources, 21, 308–313. (in Chinese)
Kang Z, Buchenauer H. 2002. Immunocytochemical localization of β-1,3-glucanase and chitinase in Fusarium culmorum-infected wheat spikes. Physiological and Molecular Plant Pathology, 60, 141–153.
Kazan K, Gardiner D M. 2018. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Molecular Plant Pathology, 19, 1547–1562.
Kim S G, Kim S T, Wang Y, Kim S K, Lee C H, Kim K K, Kim J K, Lee S Y, Kang K Y. 2010. Overexpression of rice isoflavone reductase-like gene (osirl) confers tolerance to reactive oxygen species. Physiologia Plantarum, 138, 1–9.
Kong L, Anderson J M, Ohm H W. 2005. Induction of wheat defense and stress-related genes in response to Fusarium graminearum. Genome, 48, 29–40.
Konishi H, Yamane H, Maeshima M, Komatsu S. 2004. Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling. Plant Molecular Biology, 56, 839–848.
Lamprecht S C, Marasas W F O, Hardy M B, Calitz F J. 2006. Effect of crop rotation on crown rot and the incidence of Fusarium pseudograminearum in wheat in the Western Cape, South Africa. Australasian Plant Pathology, 35, 419–426.
Lao N T, Long D, Kiang S, Coupland G, Shoue D A, Carpita N C, Kavanagh T A. 2003. Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis. Plant Molecular Biology, 53, 647–661.
Li J, Yang X, Liu X, Yu H, Du C, Li M, He D. 2017. Proteomic analysis of the compatible interaction of wheat and powdery mildew (Blumeria graminis f. sp. tritici). Plant Physiology and Biochemistry, 111, 234–243.
Li W L, Faris J D, Muthukrishnan S, Liu D J, Chen P D, Gill B S. 2001. Isolation and characterization of novel cDNA clones of acidic chitinases and β-1,3-glucanases from wheat spikes infected by Fusarium graminearum. Theoretical and Applied Genetics, 102, 353–362.
Liu C J, Ogbonnaya F C. 2015. Resistance to Fusarium crown rot in wheat and barley: A review. Plant Breeding, 134, 365–372.
Long S P, Marshall-Colon A, Zhu X G. 2015. Meeting the global food demand of the future by engineering crop photosynthesis and yield potential. Cell, 161, 56–66.
Ma J, Li H B, Zhang C Y, Yang X M, Liu Y X, Yan G J, Liu C J. 2010. Identification and validation of a major QTL conferring crown rot resistance in hexaploid wheat. Theoretical and Applied Genetics, 120, 1119–1128.
Ma J, Li R, Wang H, Li D, Wang X, Zhang Y, Zhen W, Duan H, Yan G, Li Y. 2017. Transcriptomics analyses reveal wheat responses to drought stress during reproductive stages under field conditions. Frontiers in Plant Science, 8, 592.
Ma J, Stiller J, Zhao Q, Feng Q, Cavanagh C, Wang P, Gardiner D, Choulet F, Feuillet C, Zheng Y L, Wei Y, Yan G, Han B, Manners J M, Liu C J. 2014. Transcriptome and allele specificity associated with a 3BL locus for Fusarium crown rot resistance in bread wheat. PLoS ONE, 9, e113309.
Ma J, Yan G J, Liu C J. 2012. Development of near-isogenic lines for a major QTL on 3BL conferring Fusarium crown rot resistance in hexaploid wheat. Euphytica, 183, 147–152.
Malec P, Yahalom A, Chamovitz D A. 2002. Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana. Photochemistry and Photobiology, 75, 178–183.
Martin A, Bovill W D, Percy C D, Herde D, Fletcher S, Kelly A, Neate S M, Sutherl M W. 2015. Markers for seedling and adult plant crown rot resistance in four partially resistant bread wheat sources. Theoretical and Applied Genetics, 128, 377–385.
Mishra P K, Tewari J P, Clear R M, Turkington T K. 2006. Genetic diversity and recombination within populations of Fusarium pseudograminearum from western Canada. International Microbiology: The Official Journal of the Spanish Society for Microbiology, 9, 65–68.
Murray G M, Brennan J P. 2009. Estimating disease losses to the Australian wheat industry. Australasian Plant Pathology, 38, 558–570.
Nalam V J, Alam S, Keereetaweep J, Venables B, Burdan D, Lee H, Trick H N, Sarowar S, Makandar R, Shah J. 2015. Facilitation of Fusarium graminearum infection by 9-lipoxygenases in Arabidopsis and wheat. Molecular Plant–Microbe Interactions, 28, 1142–1152.
Nishizawa Y, Nishio Z, Nakazono K, Soma M, Nakajima E, Ugaki M, Hibi T. 1999. Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase. Theoretical and Applied Genetics, 99, 383–390.
Poole G J, Smiley R W, Paulitz T C, Walker C A, Carter A H, See D R, Garland-Campbell K. 2012. Identification of quantitative trait loci (QTL) for resistance to Fusarium crown rot (Fusarium pseudograminearum) in multiple assay environments in the Pacific Northwestern US. Theoretical and Applied Genetics, 125, 91–107.
Powell J J, Carere J, Fitzgerald T L, Stiller J, Covarelli L, Xu Q, Gubler F, Colgrave M L, Gardiner D M, Manners J M, Henry R J, Kazan K. 2017. The Fusarium crown rot pathogen Fusarium pseudograminearum, triggers a suite of transcriptional and metabolic changes in bread wheat (Triticum aestivum L.). Annals of Botany, 119, 853–867.
Pritsch C, Muehlbauer G J, Bushnell W R, Somers D A, Vance C P. 2000. Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum. Molecular Plant–Microbe Interactions, 13, 159–169.
Pritsch C, Vance C P, Bushnell W R, Somers D A, Hohn T M, Muehlbauer G J. 2001. Systemic expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection. Physiological and Molecular Plant Pathology, 58, 1–12.
Qin Y X, Qin F. 2016. Dehydrins from wheat×Thinopyrum ponticum amphiploid increase salinity and drought tolerance under their own inducible promoters without growth retardation. Plant Physiology and Biochemistry, 99, 142–149.
Shin S, Mackintosh C A, Lewis J, Heinen S J, Radmer L, Dill-Macky R, Baldridge G D, Zeyen R J, Muehlbauer G J. 2008. Transgenic wheat expressing a barley class II chitinase gene has enhanced resistance against Fusarium graminearum. Journal of Experimental Botany, 59, 2371–2378.
Smiley R W, Gourlie J A, Easley S A, Patterson L M, Whittaker R G. 2005. Crop damage estimates for crown rot of wheat and barley in the pacific northwest. Plant Disease, 89, 595–604.
Summerell B A, Burgess L W. 1988. Stubble management practices and the survival of Fusarium graminearum Group 1 in wheat stubble residues. Australasian Plant Pathology, 17, 88–93.
Summerell B A, Burgess L W, Klein T A, Pattison A B. 1990. Stubble management and the site of penetration of wheat by Fusarium graminearum Group 1. Phytopathology, 80, 877–879.
Takahashi W, Fujimori M, Miura Y, Komatsu T, Nishizawa Y, Hibi T, Takamizo T. 2005. Increased resistance to crown rust disease in transgenic Italian ryegrass (Lolium multiflorum Lam.) expressing the rice chitinase gene. Plant Cell Reports, 23, 811–818.
Wallwork H, Butt M, Cheong J P E, Williams K J. 2004. Resistance to crown rot in wheat identified through an improved method for screening adult plants. Australasian Plant Pathology, 33, 1–7.
Wang N, Zhao J, He X, Sun H, Zhang G, Wu F. 2015. Comparative proteomic analysis of drought tolerance in the two contrasting Tibetan wild genotypes and cultivated genotype. BMC Genomics, 16, 432.
Whitney S M, Houtz R L, Alonso H. 2011. Advancing our understanding and capacity to engineer nature’s CO2-sequestering enzyme, RuBisCO. Plant Physiology, 155, 27–35.
Xu F, Song Y L, Zhou Y L, Zhang H, Wang J M, Li Y H, Han Z H. 2016. Occurrence dynamics and characteristics of Fusarium root and crown rot of wheat in Henan Province during 2013–2016. Plant Protection, 6, 23. (in Chinese)
Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N. 2000. Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Reports, 19, 639–646.
Yang X, Pan Y, Singh P K, He X, Ren Y, Zhao L, Zhang N, Cheng S, Chen F. 2019. Investigation and genome-wide association study for Fusarium crown rot resistance in Chinese common wheat. BMC Plant Biology, 19, 153.
Yang Y, He X L, Hu Y F, Hou Y, Niu Y J, Dai J L, Yuan H X, Li H L. 2015. Resistance of wheat cultivars in Huang-Huai region of China to crown rot caused by Fusarium pseudograminearum. Journal of Triticeae Crops, 3, 339–345. (in Chinese)
Zhang X H, Fu J M, Hiromasa Y, Pan H Y, Bai G H. 2013. Differentially expressed proteins associated with Fusarium head blight resistance in wheat. PLoS ONE, 8, e82079.
Zhang X X, Sun H Y, Shen C M, Li W, Yu H S, Chen H G. 2015. Survey of Fusarium spp. causing wheat crown rot in major winter wheat growing regions of china. Plant Disease, 99, 1610–1615. (in Chinese)
Zheng Z, Kilian A, Yan G J, Liu C J. 2014. QTL conferring Fusarium crown rot resistance in the elite bread wheat variety EGA Wylie. PLoS ONE, 9, e96011.
Zheng Z, Ma J, Stiller J, Zhao Q, Feng Q, Choulet F, Feuillet C, Zheng Y L, Wei Y, Han B, Yan G, Manners J M, Liu C J. 2015. Fine mapping of a large-effect QTL conferring Fusarium crown rot resistance on the long arm of chromosome 3B in hexaploid wheat. BMC Genomics, 16, 850.
Zhou H F, Yang Y, Niu Y J, Yang H X, Li H L. 2014. Occurrence and control methods of crown rot of wheat. Journal of Henan Agricultural Sciences, 43, 114–117. (in Chinese)
Zhu S M, Liu Q R. 2016. The cause reason and comprehensive control of wheat crown rot in Xinxiang. China Plant Protection, 36, 40–42. (in Chinese)