Cloning and Functional Study of UeCS3.1 Gene in Ustilago esculenta
WU Min*, ZHANG Ya-Fen*, XIA Wen-Qiang, HU Peng, CHEN Yue, YU Xiao-Ping, YE Zi-Hong**
Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China
Abstract：Ustilago esculenta, a basidiomycete fungus, can infect Zizania latifolia and induce the swelling of tissues near the base of the host plant. The formed edible gall called 'Jiaobai', that is the second largest aquatic vegetable in China. Chitin, the β-1,4-linked linear homopolymer of N-acetylglucosamine (GlcNAc), is the major component of the fungal cell wall and is crucial for the morphogenesis and survival of fungi. The synthesis of chitin is highly conserved and involves several enzymes, of which the chitin synthase (CS) is the key enzyme. Previous study revealed that there are at least 2 but maybe up to 20 chitin synthase genes per species, which were classified into 7 classes. They are basically involved in the whole process of fungal growth and pathogenicity. ClassⅢchitin synthases are crucial for hyphal growth and pathogenicity in some filamentous fungi. In this study, ClassⅢchitin synthases UeCS3.1 (GenBank No. KU302676) in U. esculenta was cloned based on the whole genome sequence of U. esculenta. The genomic DNA of UeCS3.1 was 3 054 bp with one intron. The full length of its ORF was 2 751 bp encoding a protein with 916 amino acids. In addition, it was predicted that UeCS3.1 had 2 catalytic domains of Chitin-synth-1(pfam01644) and Chitin-synth-1N (pfam08407), seven deduced transmembrane regions, with the theoretical isoelectric point of 8.78 and the molecular weight of 103.73 kD by the biological information analysis of protein structure. The phylogenetic analysis showed that the UeCS3.1 has the highest homology with Umchs1 of Ustilago maydis. Then qRT-PCR was used to detect the relative expression of UeCS3.1 during haploid growth and mating process. The expression analysis showed that the expression of UeCS3.1 was up-regulated during haploid growth and mating process and showed a different relative expression level in the T and MT strains. The relative expression of UeCS3.1 in MT strains was higher than that in T strains at 24~48 h during haploid growth process. Furthermore, during the mating process, the relative expression of UeCS3.1 in T strains was significantly higher than that in MT strains at 24 h and it was continuously up-regulated during 0~72 h while that in MT strains tended to be stable at 72 h. So it was speculated that UeCS3.1 was involved in the process of haploid and mating. For further verification, UeCS3.1 deletion strains were constructed by homologous recombination and PEG mediated protoplast transformation. The morphology of UeCS3.1 deletion strains was observed by microscope and growth curve of UeCS3.1 deletion strains was drawn by the values of OD600 at 12, 24, 36, 48, 60 and 72 h. The results showed that the morphology and growth curve of UeCS3.1 deletion strains did not change significantly during the haploid phase. During the mating process, the conjugation tubes of UeCS3.1 deletion strains were formed at 36 h while wild type strains were formed at 24 h. Meanwhile, the hypha of UeCS3.1 deletion strains was shorter than that of wild type strains. Thus, the ability of fusion and hyphal growth of UeCS3.1 deletion strains decreased significantly during the mating process. Totally, these results suggest that UeCS3.1 might be involved in the mating process especially for the hyphal growth in U. esculenta. Above all, this study preliminarily explored the function of the classⅢchitin synthases of U. esculenta, and discussed its role in the mating process, which provided basic materials for the pathogenic mechanism of U. esculenta.
[1] 曹乾超, 张雅芬, 崔海峰, 等. 2016. 菰黑粉菌的研究进展[J]. 长江蔬菜, (6): 25-29. (Cao Q C, Zhang Y F, Cui H F, et al. 2016. Research progress of Ustilago esculenta[J]. Journal of Changjiang Vegetable, (6): 25-29.) [2] 程岩, 沈崇尧, 裘维蕃. 1989. 关于茭白黑粉菌的正名问题[J]. 真菌学报, (1): 9-16. (Chen Y, Shen C Y, Qiu W F. 1989. On the nomenclature of Ustilago esculenta[J]. Acta Mycologica Sinica, (1): 9-16.) [3] 胡鹏. 2016. 菰黑粉菌T型和MT型菌株差异性研究及b基因功能分析[D]. 硕士学位论文,中国计量大学,导师: 叶子弘. pp. 73-77. (Hu P.2016. Analysis of differentation between T and MT strains and b genes function in Ustilago esculenta[D]. Thesis for M.S., China Jiliang University, Supervisor: Ye Z H. pp. 73-77.) [4] 俞晓平, 李建荣, 施建苗, 等. 2003. 水生蔬菜茭白及其无害化生产技术[J]. 浙江农业学报, 15(3): 0-117. (Yu X P, Li J R, Shi J M, et al.2003. Aquatic vegetable Zizania latifolia and its harmless production technology[J]. Acta Agriculturae Zhejiangensis, 15(3): 0-117.) [5] Boyce K J, Andrianopoulos A.2015. Fungal dimorphism: The switch from hyphae to yeast is a specialized morphogenetic adaptation allowing colonization of a host[J]. FEMS Microbiology Reviews, 39(6): 797-811. [6] Bueter C L, Specht C A, Levitz S M.2013. Innate sensing of chitin and chitosan[J]. PLoS Pathogens, 9(1): 3. [7] Cabib E, Roh D H, Schmidt M, et al.2001. The yeast cell wall and septum as paradigms of cell growth and morphogenesis[J]. The Journal of Biological Chemistry, 276(23): 19679-19682. [8] Choquer M, Boccara M, Goncalves I R, et al.2004. Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes[J]. European Journal of Biochemistry, 271(11): 2153-2164. [9] Choquer M, Boccara M, Vidal-Cros A.2003. A semi-quantitative RT-PCR method to readily compare expression levels within Botrytis cinerea multigenic families in vitro and in planta[J]. Current Genetics, 43(4): 303-309. [10] Din A B, Specht C A, Robbins P W, et al.1996. chs-4, a class Ⅳ chitin synthase gene from Neurospora crassa[J]. Molecular & General Genetics, 250(2): 214-22. [11] Fukuda K, Yamada K, Deoka K, et al.2009. Class Ⅲ chitin synthase ChsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development[J]. Eukaryotic Cell, 8(7): 945-956. [12] Garcerá-Teruel A, Xoconostle-Cázares B, Rosas-Quijano R, et al.2004. Loss of virulence in Ustilago maydis by Umchs6 gene disruption[J]. Research in Microbiology, 155(2): 87-97. [13] Guo H B, Li S M, Peng J, et al.2007. Zizania latifolia Turcz. cultivated in China[J]. Genetic Resources & Crop Evolution, 54(6): 1211-1217. [14] Latgé J P.2007. The cell wall: A carbohydrate armour for the fungal cell[J]. Molecular Microbiology, 66(2): 279-290. [15] Lee J I, Choi J H, Park B C, et al.2004. Differential expression of the chitin synthase genes of Aspergillus nidulans, chsA, chsB, and chsC, in response to developmental status and environmental factors[J]. Fungal Genetics and Biology, 41(6): 635-646. [16] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method[J]. Methods, 25(4): 402-408. [17] Mellado E, Aufauvre-Brown A, Gow N A, et al.1996. The Aspergillus fumigatus chsC and chsG genes encode class Ⅲ chitin synthases with different functions[J]. Molecular Microbiology, 20(3): 667-79. [18] Mellado E, Dubreucq G, Mol P, et al.2003. Cell wall biogenesis in a double chitin synthase mutant (chsG-/chsE-) of Aspergillus fumigatus[J]. Fungal Genetics and Biology, 38(1): 98-109. [19] Merzendorfer H.2011. The cellular basis of chitin synthesis in fungi and insects: Common principles and differences[J]. European Journal of Cell Biology, 90(9): 759-769. [20] Mio T, Yabe T, Sudoh M, et al.1996. Role of three chitin synthase genes in the growth of Candida albicans[J]. Journal of Bacteriology, 178(8): 2416-2419. [21] Muzzarelli R A.1999. Native, industrial and fossil chitins[J]. Experientia Supplementum, 87: 1-6. [22] Niño-Vega G A, Carrero L, San-Blas G.2004. Isolation of the CHS4 gene of Paracoccidioides brasiliensis and its accommodation in a new class of chitin synthases[J]. Medical Mycology, 42(1): 51-57. [23] Shaw J A, Mol P C, Bowers B, et al.1991. The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle[J]. The Journal of Cell Biology, 114(1): 111-123. [24] Soulié M C, Perino C, Piffeteau A, et al.2006. Botrytis cinerea virulence is drastically reduced after disruption of chitin synthase class Ⅲ gene (Bcchs3a)[J]. Cellular Microbiology, 8(8): 1310-1321. [25] Takeshita N, Ohta A, Horiuchi H.2002. csmA, a gene encoding a class V chitin synthase with a myosin motor-like domain of Aspergillus nidulans, is translated as a single polypeptide and regulated in response to osmotic conditions[J]. Biochemical and Biophysical Research Communications, 298(1): 103-109. [26] Terrell E E, Batra L R.1982. Zizania latifolia and Ustilago esculenta, a grass-fungus association[J]. Economic Botany, 36(3): 274-285. [27] Wang Z, Szaniszlo P J.2000. WdCHS3, a gene that encodes a class Ⅲ chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions[J]. Journal of Bacteriology, 182(4): 874-881. [28] Weber I, Assmann D, Thines E, et al.2006. Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis[J]. The Plant Cell, 18(1): 225-242. [29] Ye Z H, Pan Y, Zhang Y F, et al.2017. Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome[J]. DNA Research, 24(6): 635-648. [30] You W Y, Liu Q A, Zou K Q, et al.2011. Morphological and molecular differences in two strains of Ustilago esculenta[J]. Current Microbiology, 62(1): 44-54. [31] Zhang J Z, Chu F Q, Guo D P, et al.2012. Cytology and ultrastructure of interactions between Ustilago esculenta and Zizania latifolia[J]. Mycological Progress, 11(2): 499-508. [32] Zhang J Z, Chu F Q, Guo D P, et al.2014. The vacuoles containing multivesicular bodies: A new observation in interaction between Ustilago esculenta and Zizania latifolia[J]. European Journal of Plant Pathology, 138(1): 79-91. [33] Zhang Y F, Cao Q C, Hu P, et al.2017. Investigation on the differentiation of two Ustilago esculenta strains-implications of a relationship with the host phenotypes appearing in the fields[J]. BMC Microbiology, 17: 16. [34] Zhang Y F, Ge Q W, Cao Q C, et al.2018. Cloning and characterization of two MAPK genes UeKpp2 and UeKpp6 in Ustilago esculenta[J]. Current Microbiology, 75(8): 1016-1024. [35] Zhang Y F, Liu H L, Cao Q C, et al.2018. Cloning and characterization of the UePrf1 gene in Ustilago esculenta[J]. FEMS Microbiology Letters, 365(12): 10.
