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Journal of Integrative Agriculture  2021, Vol. 20 Issue (9): 2410-2423    DOI: 10.1016/S2095-3119(20)63558-4
Special Issue: 园艺-分子生物合辑Horticulture — Genetics · Breeding
Horticulture Advanced Online Publication | Current Issue | Archive | Adv Search |
Identification and expression analysis of the PbrMLO gene family in pear, and functional verification of PbrMLO23
GUO Bing-bing1, LI Jia-ming1, LIU Xing1, 2, QIAO Xin1, Musana Rwalinda FABRICE1, WANG Peng1, ZHANG Shao-ling1, WU Ju-you
1 Center of Pear Engineering Technology Research/State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P.R.China
2 School of Life Sciences, Nanjing University, Nanjing 210023, P.R.China
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摘要  

本研究共鉴定到了24个PbrMLO基因,并将其分为5个亚家族(I,II,III,IV,V),全基因组重复和分散重复促进了PbrMLO家族的扩大。此外,基因表达分析显示PbrMLO基因分布在梨的不同组织中,表明其功能不同。我们选择PbrMLO23进行进一步的功能分析。qRT-PCR表达谱分析显示PbrMLO23在花粉中有高表达。亚细胞定位分析显示PbrMLO23位于细胞膜上。当反义寡核苷酸降低PbrMLO23基因的表达量时,花粉管长度变长,说明PbrMLO23具有抑制花粉管生长的功能作用。综上所述,这些结果提供了对PbrMLO基因家族及其功能特征的深刻认识,为进一步分析PbrMLO在梨中的功能奠定了基础。




Abstract  
Mildew resistance locus O (MLO) is a plant-specific gene family that plays an important role in the growth and development of plants and their interactions with the environment.  However, the available information on this gene family in pear is limited.  Here, 24 PbrMLO genes were identified and divided into five subfamilies (I, II, III, IV and V).  Whole-genome duplication (WGD) and dispersed duplication contributed to the expansion of the PbrMLO family.  In addition, gene expression analysis revealed that PbrMLO genes were distributed in various pear tissues, suggesting their diverse functions.  We selected PbrMLO23 for further functional analysis.  Expression profile analysis by qRT-PCR showed that PbrMLO23 was highly expressed in pollen.  Subcellular localization analysis showed that PbrMLO23 was located on the plasma membrane.  When the expression level of PbrMLO23 was knocked down by using antisense oligonucleotides, pollen tube lengths increased, indicating that PbrMLO23 plays a functional role in inhibiting pollen tube growth.  In summary, these results provide evolutionary insight into PbrMLO and its functional characteristics and lay a foundation for further analysis of the functions of PbrMLO members in pear.
 
Keywords:  MLO        pear        gene expression        pollen tube  
Received: 07 April 2020   Accepted:
Fund: This work was supported by the National Natural Science Foundation of China (31772276), the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China, and the China Postdoctoral Science Foundation (2017M620213).
Corresponding Authors:  Correspondence WU Ju-you, Mobile: +86-13913835567, Tel/Fax: +86-25-84396485, E-mail: juyouwu@njau.edu.cn   
About author:  GUO Bing-bing, E-mail: guobingbing1989@126.com;

Cite this article: 

GUO Bing-bing, LI Jia-ming, LIU Xing, QIAO Xin, Musana Rwalinda FABRICE, WANG Peng, ZHANG Shao-ling, WU Ju-you. 2021. Identification and expression analysis of the PbrMLO gene family in pear, and functional verification of PbrMLO23. Journal of Integrative Agriculture, 20(9): 2410-2423.

Acevedo-Garcia J, Kusch S, Panstruga R. 2014. Magical mystery tour: MLO proteins in plant immunity and beyond. New Phytologist, 204, 273–281.
Bateman A, Coin L, Durbin R, Finn R, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer E, Studholme D J Y, Eddy S. 2004. The Pfam protein families database. Nucleic Acids Research, 32, D138–D141.
Chen J, Wang P, De Graaf B H J, Zhang H, Jiao H J, Tang C, Zhanng S L, Wu J Y. 2018. Phosphatidic acid counteracts S-RNase signaling in pollen by stabilizing the actin cytoskeleton. The Plant Cell, 30, 1023–1039.
Chen Y, Wang Y, Zhang H. 2014. Genome-wide analysis of the mildew resistance locus o (MLO) gene family in tomato (Solanum lycopersicum L.). Plant Omics, 7, 87–93.
Chen Z, Hartmann H A, Wu M J, Friedman E J, Chen J G, Pulley M, Schulze-Lefert P, Panstruga R, Jones A M. 2006. Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins. Plant Molecular Biology, 60, 583–597.
Chen Z Q, Noir S, Kwaaitaal M, H. Hartmann A, Wu M J, Mudgil Y, Sukumar P, Muday G, Panstruga R, Jones A M. 2009. Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis. The Plant Cell, 21, 1972–1991.
Devoto A, Hartmann H A, Piffanelli P, Elliott C, Simmons C, Taramino G, Goh C S, Cohen F E, Emerson B C, Schulze-Lefert P, Panstruga R. 2003. Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. Journal of Molecular Evolution, 56, 77–88.
Devoto A, Piffanelli P, Nilsson I M, Wallin E, Panstruga R, von Heijne G, Schulze-Lefert P. 1999. Topology, subcellular localization, and sequence diversity of the Mlo family in plants. Journal of Biological Chemistry, 274, 34993–35004.
Eddy S. 1998. HMMER: Profile HMMs for protein sequence analysis. Bioinformatics, 14, 755–763.
Edgar R C. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797.
Estruch J J, Kadwell S, Merlin E, Crossland L. 1994. Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proceedings of the National Academy of Sciences of the United States of America, 91, 8837–8841.
Fawcett J A, Maere S, Van De Peer Y. 2009. Plants with double genomes might have had a better chance to survive the Cretaceous-Tertiary extinction event. Proceedings of the National Academy of Sciences of the United States of America, 106, 5737–5742.
Freeling M. 2009. Bias in plant gene content following different sorts of duplication: tandem, whole-genome, segmental, or by transposition. Annual Review of Plant Biology, 60, 433–453.
De Graaf B H J, Rudd J J, Wheeler M J, Perry R M, Bell E M, Osman K, Franklin C H, Franklin-Tong V E. 2006. Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen. Nature, 444, 490–493.
Jacott C N, Charpentier M, Murray J D and Ridout C J. 2020. Mildew Locus O facilitates colonization by arbuscular mycorrhizal fungi in angiosperms. New Phytologist, 227, 343–351.
Jørgensen I H. 1992. Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica, 63, 141–152.
Hall T A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Hepler P K, Vidali L, Cheung A Y. 2001. Polarized cell growth in higher plants. Annual Review of Cell and Developmental Biology, 17, 159–187.
Iovieno P, Bracuto V, Pavan S, Lotti C, Ricciardi L, Andolfo G. 2016. Identification and functional inference on the Mlo family in viridiplantae. Journal of Plant Pathology, 98, 587–594.
Kessler S A, Shimosato-Asano H, Keinath N F, Wuest S E, Ingram G, Panstruga R, Grossniklaus U. 2010. Conserved molecular components for pollen tube reception and fungal invasion. Science, 330, 968–971.
Kim M C, Lee S H, Kim J K, Chun H J, Choi M S, Chung W S, Moon B C, Kang C H, Park C Y, YOO J H, Kanng Y H, Koo S C, Koo Y D, Jung J C, Kin S T, Schulze-Lefert P, Lee S Y, Cho M J. 2002a. MLO, a modulator of plant defense and cell death, is a novel calmodulin-binding protein isolation and characterization of a rice Mlo homologue. Journal of Biological Chemistry, 277, 19304–19314.
Kim M C, Panstruga R, Elliott C, Müller J, Devoto A, Yoon H W, Park H C, Cho M J, Schulze-Lefert P. 2002b. Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature, 416, 447.
Konishi S, Sasakuma T, Sasanuma T. 2010. Identification of novel Mlo family members in wheat and their genetic characterization. Genes & Genetic Systems, 85, 167–175.
Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874.
Li C L, Meng D, Miguel A. Piñeros, Mao Y X, Abhaya M. Dandekar, Cheng L L. 2020a. A sugar transporter takes up both hexose and sucrose for sorbitol-modulated in vitro pollen tube growth in apple. The Plant Cell, 32, 449–469.
Liu L P, Qu J W, Yi X Q, Huang H H. 2017. Genome-wide identi?cation, classi?cation and expression analysis of the mildew resistance locus O (MLO) gene family in sweet orange (Citrus sinensis). Brazilian Archives of Biology and Technology, 60, e17160474..
Liu Q, Zhu H. 2008. Molecular evolution of the MLO gene family in Oryza sativa and their functional divergence. Gene, 409, 1–10.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods, 25, 402–408.
Lynch M, Conery J S. 2000. The evolutionary fate and consequences of duplicate genes. Science, 290, 1151–1155.
Malik C P, Chhabra N. 1976. Hormonal regulation of pollen germination and pollen tube elongation inArachis hypogea Reitz. Proceedings of the Indian Academy of Sciences (Section B), 84, 101–108.
Meng J G, Liang L, Jia P F, Wang Y C, Li H J, Yang W C. 2020. Integration of ovular signals and exocytosis of a Ca2+ channel by MLOs in pollen tube guidance. Nature Plants, 6, 143–145.
Meurer-Grimes B. 1995. New evidence for the systematic significance of acylated spermidines and flavonoids in pollen of Higher Hamamelidae. Brittonia, 47, 130–142.
Moutinho A, Hussey P J, Trewavas A J, Malho R. 2001. cAMP acts as a second messenger in pollen tube growth and reorientation. Proceedings of the National Academy of Sciences of the United States of America, 98, 10481–10486.
Panstruga R. 2005. Serpentine plant MLO proteins as entry portals for powdery mildew fungi. Biochemical Society Transactions, 33, 389–392.
Pavan S, Jacobsen E, Visser R G F, Bai Y. 2010. Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance. Molecular Breeding, 25, 1–12.
Pessina S, Pavan S, Catalano D, Gallotta A, Visser R G, Bai Y, Malnoy M, Schouten H J. 2014. Characterization of the MLO gene family in Rosaceae and gene expression analysis in Malus domestica. BMC Genomics, 15, 618.
Pleskot R, Potocký M, Pejchar P, Linek J, Bezvoda R, Martinec J, Valentová O, Novotná Z, ?árský V. 2010. Mutual regulation of plant phospholipase D and the actin cytoskeleton. The Plant Journal, 62, 494–507.
Polanco C, de Miera L E S, Bett K, de la Vega M P. 2018. A genome-wide identification and comparative analysis of the lentil MLO genes. PLoS ONE, 13, e0194945.
Qiao X, Li M, Li L, Yin H, Wu J, Zhang S L. 2015. Genome-wide identification and comparative analysis of the heat shock transcription factor family in Chinese white pear (Pyrus bretschneideri) and five other Rosaceae species. BMC Plant Biology, 15, 12.
Rispail N, Rubiales D. 2016. Genome-wide identification and comparison of legume MLO gene family. Scientific Reports, 6, 32673.
Tang H, Wang X, Bowers J E, Ming R, Alam M, Paterson A H. 2008. Unraveling ancient hexaploidy through multiply-aligned angiosperm gene maps. Genome Research, 18, 1944–1954.
Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673–4680.
Thussagunpanit J, Jutamanee K, Chai-arree W, Kaveeta L. 2012. Increasing photosynthetic efficiency and pollen germination with 24-Epibrassinolide in rice (Oryza sativa L.) under heat stress. Thai Juornal of Botany, 4, 135–143.
Vogler F, Schmalzl C, Englhart M, Bircheneder M, Sprunck S. 2014. Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reproduction, 27, 153–167.
Wang C L, Xu G H, Jiang X T, Chen G, Wu J, Wu H Q, Zhang S L. 2009. S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro. The Plant Journal, 57, 220–229.
Wang N, Xiang Y, Fang L C, Wang Y J, Xin H P, Li S H. 2013. Patterns of gene duplication and their contribution to expansion of gene families in grapevine. Plant Molecular Biology Reporter, 31, 852–861.
Wang Y, Tang H, DeBarry J D, Tan X, Li J, Wang X, Lee T, Jin H Z, Marler B, Guo H, Kissinger J C, Paterson A H. 2012.MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Research, 40, e49-e49.
Wang Y P, Wang X Y, Tang H B, Tan X, Ficklin S P, Feltus F A, Paterson A H. 2011. Modes of gene duplication contribute differently to genetic novelty and redundancy, but show parallels across divergent angiosperms. PLoS One, 6, e28150.
Wu J, Wang Z, Shi Z, Zhanng S, Ming R, Zhu S L, Khan H A, Tao S T, Korban S S, Wang H, Chen N J, Nishio T, Xu X, Cong L, Qi K J, Huang X S, Wang Y T, Zhao X, Wu J Y, Deng C, et al. 2013. The genome of the pear (Pyrus bretschneideri Rehd.) Genome Research, 23, 396–408.
Wu J Y, Qin X Y, Tao S T, Jiang X T, Liang Y K, Zhang S L. 2014. Long-chain base phosphates modulate pollen tube growth via channel-mediated influx of calcium. The Plant Journal, 79, 507–516.
Wu J Y, Shang Z L, Wu J, Jiang X T, Moschou P N, Sun W, Roubelakis-Angelakis K, Zhang S L. 2010. Spermidine oxidase-derived H2O2 regulates pollen plasma membrane hyperpolarization-activated Ca2+-permeable channels and pollen tube growth. The Plant Journal, 63, 1042–1053.
Wu J Z, Lin Y, Zhang X L, Pang D W, Zhao J. 2008a. IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri. Journal of Experimental Botany, 59, 2529–2543.
Wu J Z, Qin Y, Zhao J. 2008b. Pollen tube growth is affected by exogenous hormones and correlated with hormone changes in styles in Torenia fournieri L. Plant Growth Regulation, 55, 137–148.
Yi J, An S, An G. 2014. OsMLO12, encoding seven transmembrane proteins, is involved with pollen hydration in rice. Plant Reproduction, 27, 169–180.
Zhang J. 2003. Evolution by gene duplication: An update. Trends in Ecology & Evolution, 18, 292–298.
Zheng Z, Appiano M, Pavan S, Bracuto V, Ricciardi L, Visser R G F, Wolters A M A, Bai Y. 2016. Genome-wide study of the tomato SlMLO gene family and its functional characterization in response to the powdery mildew fungus Oidiumneo lycopersici. Frontiers in Plant Science, 7, 380.
Zhou S J, Jing Z, Shi J L. 2013. Genome-wide identification, characterization, and expression analysis of the MLO gene family in Cucumis sativus. Genetics and Molecular Research, 12, 6565–6578.
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