Interaction mechanism between OsCdc48 and Pik1-H4, and its regulation on rice blast disease resistance
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摘要:目的
探究Pik1-H4与细胞周期蛋白OsCdc48的互作机制，明确其在稻瘟病抗性中的作用。
方法首先，分别利用酵母双杂交试验和荧光素酶互补试验验证Pik1-H4与OsCdc48的相互作用；然后，通过RT-qPCR分析OsCdc48在稻瘟病菌侵染后的表达情况及组织表达特异性；其次，分析OsCdc48的序列保守性、蛋白结构域、系统进化关系、蛋白质三维结构预测及其亚细胞定位；最后，利用CRISPR/Cas9创制OsCdc48突变体，并对其进行稻瘟病抗性鉴定和病程相关基因表达分析。
结果证实了Pik1-H4与OsCdc48的互作，且OsCdc48受稻瘟病菌侵染诱导表达。OsCdc48在各组织中均有表达，其编码蛋白定位于细胞核与细胞质。OsCdc48在不同物种中序列保守，与玉米和高粱的亲缘关系最近，预测会形成同源六聚体。OsCdc48功能缺失突变体ko-oscdc48病程相关基因上调表达，对稻瘟病抗性增强。
结论本研究为深入揭示OsCdc48与NLR蛋白Pik1-H4调控稻瘟病抗性的机制及水稻抗病育种提供了理论基础。
Abstract:ObjectiveTo investigate the interaction mechanism between Pik1-H4 and cell division cycle protein OsCdc48, clarify its role in rice blast disease resistance.
MethodFirstly, the interactions between Pik1-H4 and OsCdc48 were verified using yeast two-hybrid and luciferase complementation assays. Then, the expression patterns of OsCdc48 after infection of Magnaporthe oryzae and its tissue-specific expression were analyzed by RT-qPCR. Next, the determination of OsCdc48’s sequence conservation, protein domains, phylogenetic relationships, protein 3D structure prediction and subcellular localization were performed. Finally, OsCdc48 mutant was created using CRISPR/Cas9 technology, the resistance identification of transgenic mutant to rice blast disease and expression analysis of pathogenesis-related genes were also conducted.
ResultThe interactions between Pik1-H4 and OsCdc48 were confirmed, and OsCdc48 was induced by rice blast fungus infection. OsCdc48 was expressed in all tissues and was localized to the nucleus and cytoplasm. The sequence of OsCdc48 was conserved across different species, with the closest phylogenetic relationship to maize and sorghum, and it might form homologous hexamers. The OsCdc48 loss-of-function mutant ko-oscdc48 up-regulated the expression of disease-related genes and enhanced the resistance to rice blast disease.
ConclusionThis study lays a theoretical basis for further elucidating the mechanism of OsCdc48 and NLR protein Pik1-H4 regulating blast disease resistance and rice disease-resistant breeding.
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Keywords:
- Oryza sativa L. /
- Rice blast disease /
- Magnaporthe oryzae /
- Pik1-H4 /
- OsCdc48
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图 3 稻瘟病菌侵染过程中OsCdc48的表达分析
图中数据是3次独立试验的平均值±标准差，各图中柱子上方的不同小写字母表示在P < 0.05水平差异显著(单因素方差分析)。
Figure 3. Analysis of OsCdc48 expression during infection with Magnaporthe oryzae
Data presented in two figures are means ± standard deviations of three independent experiments, different lowercase letters above the columns in each figure indicate significant differences at P < 0.05 (One-way ANOVA).
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图 4 OsCdc48序列分析
图C中，黑色圆点为OsCdc48；图D中，第1个序列为OsCdc48，第2个序列为AtCdc48。
Figure 4. Sequence analysis of OsCdc48
In figure C, the black dot represents OsCdc48; In figure D, the first sequence represents OsCdc48, the second sequence represents AtCdc48.
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图 6 OsCdc48在水稻各组织中的相对表达量
图中数据是3次独立试验的平均值±标准差，柱子上方的不同小写字母表示在P < 0.05水平差异显著(单因素方差分析)。
Figure 6. Relative expression of OsCdc48 in various rice tissues
Data presented in figure are means ± standard deviations of three independent experiments, different lowercase letters above the columns indicate significant differences at P < 0.05 (One-way ANOVA).
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图 7 ko-oscdc48突变体的靶位点和突变序列
Figure 7. Target sites and mutation sequences of ko-oscdc48 mutant
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图 9 接种GUY11和GDYJ7后ko-oscdc48与野生型Pik-H4 NIL的表型
Figure 9. Phenotype of ko-oscdc48 compared to the wild type Pik-H4 NIL after inoculation with GUY11 and GDYJ7
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图 10 接种GUY11和GDYJ7后ko-oscdc48与野生型Pik-H4 NIL的病斑长度
图中数据为平均值±标准差，n > 6，**表示突变体与野生型在P < 0.01水平差异显著(t检验)。
Figure 10. Lesion length of ko-oscdc48 compared to the wild type Pik-H4 NIL after inoculation with GUY11 and GDYJ7
Data presented in two figures are means ± standard deviations, n > 6, ** indicates significant differences between the mutant and wild type at P < 0.01 level (t test).
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图 11 接种GUY11和GDYJ7后ko-oscdc48与野生型Pik-H4 NIL病程相关基因的表达
图中数据是3次独立试验的平均值±标准差，*和***分别表示在P < 0.05和P < 0.001水平差异显著(t检验)。
Figure 11. Expression of disease-related genes in ko-oscdc48 compared to the wild type Pik-H4 NIL after inoculation with GUY11 and GDYJ7
Data presented in three figures are means ± standard deviations of three independent experiments, * and *** indicate significant differences at P < 0.05 and P < 0.001 levels respectively (t test).
表 1 本研究用到的引物
Table 1 Primers used in this study
名称
Name正向序列(5′→3′)
Forward sequence反向序列(5′→3′)
Reverse sequenceOsCdc48-target TAGGTCTCCTTATGGACCCCC GTTTTAGAGCTAGAA CGGGTCTCAATAAAGCAGTATTGCACCAGCCGGGAA OsCdc48-test AGACCTTTTCCTTGTTAGGGG AGCTTTGAGCCCATCCATAAG OsActin TGTATGCCAGTGGTCGTACCA CCAGCAAGGTCGAGACGAA OsCdc48-qPCR GAATGCTCTTGCCAAATACACC TCCTCCGCTTCTCCATCTCG PR10 CGCCGCAAGTCATGTCCTA GCTTCGTCTCCGTCGAGTGT OsPAL1 AGGAGCTCGGCTGCGTATT ATGCCGAGGAACACCTTGTT PR1a GGAAGTACGGCGAGAACATC TGGTCGTACCACTGCTTCTC OsCdc48-AAA2-CLuc agaacacgggggacgagctcATGTCCAAAGGTGTTCTGTT atcgagtacgcggaccggccATCAGGCAGAGGAATGTAGA OsCdc48-AAA1-CLuc agaacacgggggacgagctcCCTCCAAAGGGCATACTGCTT atcgagtacgcggaccggccCATCAGGAACACCAATGTCAA AD-OsCdc48-AAA2 taccagattacgctcatatgATGTCCAAAGGTGTTCTGTT gctcgagctcgatggatcccCATCAGGAACACCAATGTCAA AD-OsCdc48-AAA1 taccagattacgctcatatgCCTCCAAAGGGCATACTGCTT gctcgagctcgatggatcccTTTAGGCTTGAAGGTAAGCC Pik1 H4-NLuc ggacgagctcggtacccATGGAGGCGGCTGCCATG gtacgagatctggtcgacGCTAGTAGTTTCTGTTTGAATTTCAAT BD-Pik1 H4 gaggaggacctgcatATGATGGAGGCGGCTGCCATG gcaggtcgacggatccctaGCTAGTAGTTTCTGTTTGAATTTCAAT 
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