Identification of Rice Genes Responding to Both the Nematode Effector MgMO237 and Its Interacting Protein OsCRRSP55

doi:10.13560/j.cnki.biotech.bull.1985.2021-0508

Abstract

Abstract:

Previous studies showed that the Meloidogyne graminicola effector MgMO237 may interact with the rice protein OsCRRSP55 and inhibit rice defense responses against M. graminicola. Plant protein CRRSPs are involved in plant responses to both biotic and abiotic stresses. Understanding the influence of the interaction between MgMO237 and OsCRRSP55 on the expressions of their downstream genes will provide a theoretical basis for elucidating the mechanism of rice defense responses against root-knot nematodes. Bioinformatics analysis indicated that the OsCRRSP55 protein contained an N-terminal signal peptide and two conserved domains called the domain unknown function 26（DUF26）,in which occurred a conserved motif C-X8-C-X2-C,but had no transmembrane domains. RT-qPCR analysis indicated that the OsCRRSP55 expressed in the rice roots and it was significantly up-regulated in the galls at 7 d infection by M. graminicola compared with that in healthy root tip. The expression levels of OsCRRSP55 and its promoter binding transcription factor OsWRKY47 significantly increased in the rice treated with salicylic acid and methyl jasmonate compared with those in wild-type ones. Transcriptome sequencing showed that a jasmonic acid-responsive gene OsERF87 was down-regulated in MgMO237-transgenic rice,but up-regulated in rice overexpressing OsCRRSP55 by RT-qPCR,showing the OsERF87 was co-regulated by OsCRRSP55 and MgMO237. In sum,these results demonstrate that the M. graminicola effector MgMO237 may affect the jasmonic acid signal transmission by interacting with OsCRRSP55,which results in the suppression of plant defense responses to M. graminicola.

Key words: Meloidogyne graminicola, MgMO237, OsCRRSP55, OsERF87, jasmonate signaling pathway

LI Zhi-wen, LIU Pei-yan, CHEN Jian-song, LIAO Jin-ling, LIN Bo-rong, ZHUO Kan. Identification of Rice Genes Responding to Both the Nematode Effector MgMO237 and Its Interacting Protein OsCRRSP55[J]. Biotechnology Bulletin, 2021, 37(7): 88-97.

Figures/Tables 8

Table 1 Primers used in this study

引物 Primer	引物序列 Sequence（5'-3'）	用途 Purpose
OsCRRSP55 F	ATGGCATTCAGTAGCAAAGC	用于OsCRRSP55基因全长扩增
OsCRRSP55 R	TTAGCGGTGCACAACGATCT	用于OsCRRSP55基因全长扩增
qOsCRRSP55 F	AGGATCAGTACACGCCGTTC	用于OsCRRSP55基因的RT-qPCR检测
qOsCRRSP55 R	CGCAGTATTGACCGAACCTT	用于OsCRRSP55基因的RT-qPCR检测
OsUBQ-F	CCAGTAAGTCCTCAGCCATGGAG	用于水稻看家基因OsUBQ（Os03g13170）的RT-qPCR检测^[10]
OsUBQ-R	GGACACAATGATTAGGGATC	用于水稻看家基因OsUBQ（Os03g13170）的RT-qPCR检测^[10]
MgMO237-qPCR-F	TTGAGCGAAAGAGTCTAAATC	用于MgMO237基因的RT-qPCR检测^[10]
MgMO237-qPCR-R	ACAGCAGGTCCATACATAA	用于MgMO237基因的RT-qPCR检测^[10]
OsCRRSP55-Nco1 F	CGCGGATCCATGGCATTCAGTAGCAAAGC	用于OsCRRSP55基因瞬时表达载体构建
OsCRRSP55：FLAG-Pml1 R	CGACGCGTTTACTTATCGTCGTCATCCTTGT- AATCGCGGTGCACAACGATCTTGG	用于OsCRRSP55基因瞬时表达载体构建
OsWRKY47 F	ATGGACGACCTGCTCGAGAT	用于OsWRKY47基因的RT-qPCR检测
OsWRKY47 R	GGCGATGTCCCATGTAGCAT	用于OsWRKY47基因的RT-qPCR检测
ERF87-qPCR-F	ACTTGTCTTCTTGTTGTTGTT	用于植物激素通路相关基因的RT-qPCR检测
ERF87-qPCR-R	TCCATCTATCTACTCCTCTATCTA
PR-1b-qPCR-F	ACGGGCGTACGTACTGGCTA
PR-1b-qPCR-R	CTCGGTATGGACCGTGAAG
PR-1a- qPCR-F	CAGTGGTACGACCACGACAG
PR-1a-qPCR-R	GGCGAGTAGTTGCAGGTGAT
OsTIFY11e-qPCR-F	GTGAGGATGCTTATGCTTGC
OsTIFY11e-qPCR-R	GATCCCTAGCATATGTACTAC
IAA24-qPCR-F	AATGTTCATCTCTTCCTG
IAA24-qPCR-R	ATTCATCATCCATCTTCTC
PIL13-qPCR-F	TTCATAACCTGTCAGAGA
PIL13-qPCR-R	CGATTGCTTCATCTAATATAG
CYCLIN-D3-1-qPCR-F	ATTTCTCAACATTCCCTA
CYCLIN-D3-1-qPCR-R	ATTTCGCTATGAACATTG

Fig. 1 Multiple sequence alignment and conserved motif analysis of plant CRRSP55 proteinsSbCRRSP55（XP_0024654665.1）：Sorghum bicolor CRRSP55 protein. ZmCRRSP55（PWZ56059.1）：Zea mays CRRSP55 protein. DoCRRSP55（OEL36740.1）：Dichanthelium oligosanthes CRRSP55 protein. OsCRRSP5：Rice CRRSP55 protein. AtCRRSP55（AB020745.1 65306-64515）：Arabidopsis thaliana CRRSP55 protein. The red dotted box refers to the signal peptide sequence of rice OsCRRSP55 protein. Underlined in red refers to Unknown Function 26（DUF26）domain,and the green box refers to a conserved motif

Fig. 2 Expression levels of OsCRRSP55 gene in riceA：Expression levels of OsCRRSP55 gene in the seeds,roots,stems and leaves of 14-day-old rice. B：Expression levels of OsCRRSP55 gene in the galls at 7 d after M. graminicola infection. The error line in the figure refers to the standard deviation,*：P<0.05 significant difference. The same below

Fig. 3 Expression variations of OsCRRSP55 and WRKY47 genes in the rice treated with salicylic acid and methyl jasmonateA：Expression variations of WRKY47 and OsCRRSP55 genes in the rice treated with salicylic acid for 24 h. B：Expression variations of WRKY47 and OsCRRSP55 genes in the rice treated with methyl jasmonate for 24 h. CK：Water treatment. SA：Salicylic acid treatment. MeJA：Methyl jasmonate treatment. **：P<0.01 significant difference. The same below

Fig. 4 Identification of MgMO237-transgenic rice WT：Wild-type rice. OE61-1 and OE61-2：Different plants of the OE61 line of MgMO237-transgenic rice. OE26-1 and OE26-2：Different plants of line the OE26 of MgMO237-transgenic rice. The transcript of MgMO237 was detected in lines OE61 and OE26 but not in WT

Fig. 5 Statistics of differentially expressed genes between MgMO237-transgenic rice and wild-type rice A：Statistical diagram showing the number of differentially expressed genes in lines OE26 and OE61 of MgMO237-transgenic rice and wild-type rice lines. B：Number of up-regulated genes（left）and down-regulated genes（right）in line OE26 and OE61 of MgMO237-transgenic rice compared with wild-type rice plants. OE26 and OE61：Line OE26 and OE61 of MgMO237 transgenic rice. WT：Wild-type rice

Fig. 6 Confirmation of differentially expressed genes within plant hormone pathways in MgMO237-transgenic rice and wild-type rice by RT-qPCR OE26 and OE61：Line OE26 and OE61 of MgMO237-transgenic rice. WT：Wild-type rice

Fig. 7 Expression levels of genes within rice hormone pathways responding to OsCRRSP55 A：The expression of OsCRRSP55 was detected in rice protoplasts by Western blot. CRRSP55-1,CRRSP55-2 and CRRSP55-3 refer to three biological replicates. B：Expression levels of genes OSERF87,IAA24,PIL13,Cyclin-D3-1,PR-1b and TIFY11e in rice hormone pathways. Black columns：Protoplasts of wild-type rice. Gray columns：Rice protoplasts overexpressing OsCRRSP55

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