Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (4): 733-743.doi: 10.3864/j.issn.0578-1752.2021.04.006

• PLANT PROTECTION • Previous Articles     Next Articles

The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica

LI TianCong1(),ZHU Hang1(),WEI Ning1,LONG Feng1,WU JianYing1,ZHANG Yan1,DONG JinGao2,3,SHEN Shen1(),HAO ZhiMin1,2()   

  1. 1College of Life Sciences/Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Hebei Agricultural University, Baoding 071001, Hebei
    2State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei
    3College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
  • Received:2020-05-12 Accepted:2020-05-28 Online:2021-02-16 Published:2021-02-16
  • Contact: Shen SHEN,ZhiMin HAO E-mail:905351819@qq.com;1175593328@qq.com;shenshen0428@163.com;haozhimin@hebau.edu.cn

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Abstract:

【Objective】The objective of this study is to obtain the homologous genes of the splicing factor SC35 in Setosphaeria turcica, and to analyze the interaction among them and the expression profiles during the different growth and development stages and infection process of the pathogen. It would lay the foundation for illustrating the relationship between the SC35 family members and fungal pathogenicity.【Method】Based on amino acid sequences of SC35 protein in Arabidopsis thaliana as probe sequences, online Blastp alignment was carried out in the S. turcica genome database to obtain candidate SC35 homologues. Then they were analyzed for conserved domain and phylogenetic relationship through bioinformatics procedures. The materials of S. turcica were collected at different infection stages on maize leaves and multiple developmental stages, such as hyphae, conidia, germ tubes, appressorium and penetration hyphae, to analyze the transcription levels of SC35 homologues through real-time quantitative PCR (qRT-PCR). And their interactions were verified in vitro by the yeast two-hybrid test.【Result】Eight SC35 genes of S. turcica were obtained, named as StSC1, StSC2, StSC3, StSC4, StSC5, StSC6, StSC7, and StSC8, respectively. All of them owned typical SR protein domains, besides StSC1 showed two RRM domains. They located at different physical locations of the genome and had no linkage. Phylogenetic analysis showed that the eight alternative splicing factors were distributed in different clades, with low homology. In the process of infection on maize leaves, gene StSC1, StSC2, StSC3, StSC4, StSC5, StSC6 and StSC8 were up-regulated, while StSC7 was down-regulated at 18 h after inoculation. StSC4 showed high expression activity at 6-18 h. In different development periods, the expression levels of StSC1 were extremely significant up-regulated (P＜0.001) in the appressorium and the formation of penetration hyphae, which were 24.44 and 8.25 times higher than those in the conidial period, but the others were down-regulated during the development. The yeast two-hybrid proved that StSC4 interacted with StSC6, StSC3 with StSC8, StSC3 with StSC4, and StSC8 with StSC4. 【Conclusion】The expression patterns of SC35 are different in the infection process and multiple developmental stages of S. turcica. StSC4 is actively expressed throughout the infection process. StSC1, StSC4 and StSC6 play important regulatory roles during the formation of appressorium and penetration hyphae. StSC4 and StSC6, StSC3 and StSC8, StSC3 and StSC4, StSC8 and StSC4 interact to regulate the formation of splicing complexes.

Key words: Setosphaeria turcica, alternative splicing factor, qRT-RCR, yeast two-hybrid

Table 1

Primer sequences used for qRT-PCR"

引物Primer 序列Sequence (5′-3′)
Tubulin-F/R GGGAACTCCTCACGGATGTTG/TAACAACTGGGCAAAGGGTCA
StSC1-F/R AGAGCCAGAAACACTGAGCC/AAGCGAGAAAAGACACGCCT
StSC2-F/R GAAAATCAAGACGACCCGCC/CTTTGCCCATTGCTTTCGCT
StSC3-F/R AGCGCAAGGAGAAAGAGAGG/CCGTCTTCCTTGATACGCCT
StSC4-F/R CACTGGAGATGGTTGGCGAT/TTGCACACAAACAGCACGTT
StSC5-F/R GAACCTCTTCGTCACAGGCA/GCATGATGTTGCACTGCTCC
StSC6-F/R GCTTTGGCTTTGTCCGCTAC/ACGCGGATTCTACGTCCATC
StSC7-F/R GCGACTTTGGCACCGTTAAG/TCGAGCGTGGCATACTCAAT
StSC8-F/R GGCCTTGACCAAGAACGTCA/GCGGACATCCTTGATGACAC

Table 2

Primers used for yeast two-hybrid test"

引物Primer 序列Sequence (5′-3′)
StSC4-F/R GGCCATGGAGGCCAGTGAATTCATGTCTGTGCTTCTAGAAACATCAC/TTCATCTGCAGCTCGAGCTCGATGGATCCTCATCGTCGACGGTCCCGGT
StSC6-F/R CATATGGCCATGGAGGCCAGTGAATTCATGTCGAAACTCTTCATTG/CATCTGCAGCTCGAGCTCGATGGATCCCTACCATTGCTGCTG
StSC8-F/R TATGGCCATGGAGGCCGAATTCATGCCGTCCCGTTCGC/CTAGTTATGCGGCCGCTGCAGGTCGACGGATCCTCATCTGCGCTCCCTACTTG
StSC3-F/R ATATGGCCATGGAGGCCGAATTCATGAACTCGATCCGCGCCATCCAGGAG/GTTATGCGGCCGCTGCAGGTCGACGGATCCTTACACGCGAGTGTCTCTCT
StSC7-F/R CATATGGCCATGGAGGCCGAATTCATGGAGGACGACGCCAAGATG/TTATGCGGCCGCTGCAGGTCGACGGATCCCTACTCGTCGTCCATATCGTC

Table 3

Protein information of SC35 in S. turcica"

名称
Name		 JGI标号
ID in JGI		 NCBI登录号
Accession number in NCBI		 大小
Size (aa)		 基因长度
Gene length (bp)		 基因定位
Gene mapping
StSC1 46444 XP_008023357.1 391 1505 scaffold_14:633653-635474 (+)
StSC2 21139 XP_008027986.1 282 1031 scaffold_4:463120-464150 (-)
StSC3 171745 XP_008027251.1 304 915 scaffold_3:679211-680616 (-)
StSC4 113046 XP_008020610.1 477 1555 scaffold_1:618088-619811 (+)
StSC5 99605 XP_008030866.1 111 336 scaffold_8:693429-693764 (+)
StSC6 168126 XP_008023544.1 161 835 scaffold_14:724868-726377 (-)
StSC7 88128 XP_008025196.1 159 537 scaffold_2:109463-109999 (-)
StSC8 156630 XP_008029952.1 343 1157 scaffold_6:1923760-1924916 (+)

Fig. 1

Schematic diagram of protein structure of SC35 in S. turcica"

Fig. 2

Phylogenetic analysis of splicing factor SC35 of S. turcica and other fungi"

Fig. 3

Expression of alternative splicing factor at different developmental stages ***: P＜0.001"

Fig. 4

Expression of alternative splicing factor after inoculation at different times ***: P＜0.001"

Fig. 5

Enzyme digestion verification of two-hybrid vectors 1: StSC4-BD; 2: StSC6-BD; 3: StSC8-BD; 4: StSC3-BD; 5: StSC7-BD"

Fig. 6

Verification of the alternative splicing factor interaction through yeast two-hybrid A：8个剪接因子间相互作用关系 The interaction of 8 alternative splicing factors;B：StSC4-AD/BD与StSC6-AD/BD StSC4-AD/BD and StSC6-AD/BD; C：StSC3-AD/BD与StSC8-AD/BD StSC3-AD/BD and StSC8-AD/BD;D：StSC3-AD/BD与StSC4-AD/BD StSC3-AD/BD and StSC4-AD/BD; E：StSC8-AD/BD与StSC4-AD/BD StSC8-AD/BD and StSC4-AD/BD ○：未验证Not verified;√：互作Interacted;×：不互作Not interacted"

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