Different Reactions of Rice Monogenic Line IRBL9-W Harboring Pi9 Gene to Magnaporthe oryzae Containing AvrPi9 During Seedling and Adult-plant Stages

doi:10.16819/j.1001-7216.2021.0717

Chinese Journal OF Rice Science ›› 2021, Vol. 35 ›› Issue (3): 303-310.DOI: 10.16819/j.1001-7216.2021.0717

• Research Papers • Previous Articles

Different Reactions of Rice Monogenic Line IRBL9-W Harboring Pi9 Gene to Magnaporthe oryzae Containing AvrPi9 During Seedling and Adult-plant Stages

Shufang LIU^1,²^,^#, Liying DONG^1,²^,^#, Xundong LI^1,², Wumin ZHOU^1,³, Qinzhong YANG^1,^2,^*()

¹Agricultural Environment Resources Research Institute, Yunnan Academy of Agricultural Sciences/Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests of Yunnan Province, Kunming 650205, China
²Scientific Observing and Experimental Station of Crop Pests in Kunming, Ministry of Agriculture and Rural Affairs, Kunming 650205, China
³College of Agriculture, Yunnan University, Kunming 650091, China

Received:2020-07-24 Revised:2020-11-24 Online:2021-05-10 Published:2021-05-10
Contact: Qinzhong YANG
About author:
^#These authors contributed equally to this work;

持有Pi9基因的水稻单基因系IRBL9-W对稻瘟病菌苗期和成株期抗性差异

刘树芳^1,²^,^#, 董丽英^1,²^,^#, 李迅东^1,², 周伍民^1,³, 杨勤忠^1,^2,^*()

¹云南省农业科学院农业环境资源研究所/云南省农业跨境有害生物绿色防控重点实验室,昆明 650205
²农业农村部昆明作物有害生物科学观测实验站,昆明 650205
³云南大学农学院,昆明 650091

通讯作者: 杨勤忠
作者简介:
^#共同第一作者;
基金资助:
云南省基础研究计划重点资助项目(202001AS070006);科技部国家重点研发专项(2016YFD0100101);国家自然科学基金资助项目(31860524);云南省农业科学院院基金资助项目(YJZ201803)

Abstract

Abstract:

【Objective】 The Pi9 gene confers broad-spectrum resistance against Magnaporthe oryzae. Rice monogenic line IRBL9-W carrying single blast resistance gene Pi9 is highly resistant to leaf blast during the seedling stage, but it is susceptible to neck blast at adult-plant stage in blast nursery. It is important to analyze the mechanism behind the reversion of resistance to Magnaporthe oryzae, which will provide important information for effective utilization of Pi9 in disease-resistant rice breeding program.【Method】Eight single spore strains of M. oryzae isolated from neck blast samples of diseased IRBL9-W, and one isolate Y363 were artificially inoculated by spraying at seedling stage to pathotype their pathogenicity on 24 monogenic lines including IRBL9-W. Two out of eight strains and control strain Y363 were further selected and inoculated on IRBL9-W at late-booting stage by injecting to evaluate their pathogenicity. The alleles of M. oryzae AvrPi9 gene cognate to rice blast resistance gene Pi9 were amplified and sequenced with AvrPi9-specific primers in nine strains. Total RNA of the rice monogenic line IRBL9-W in seedling and late-booting stage was extracted from young leaf and panicle, respectively. The expression analysis of Pi9 gene was performed by semi-quantitative RT-PCR and real-time qRT-PCR. 【Result】 The monogenic line IRBL9-W was completely resistant to the nine M. oryzae strains at the seedling stage exposed to the artificial spraying. However, IRBL9-W was susceptible to panicle blast when inoculated with both two randomly selected strains (YX2-7-1 and YX2-15-1) isolated from panicle blast lesions of IRBL9-W, and the control strain Y363 by injecting inoculation at the late-booting stage. Compared with AvrPi9, allelic sequence amplified from Y363 was identical to cloned AvrPi9, and the coding region of the other eight strains isolated from IRBL9-W panicle blast was identical to AvrPi9 gene, except for a 16-bp deletion at -264 bp upstream of the coding starting position in the eight strains. Given high resistance of IRBL9-W to these strains at the seedling stage, we contended that this 16-bp deletion did not affect the function of AvrPi9 gene; qRT-PCR results showed that the relative expression level of Pi9 gene in panicle was only 47.3% as compared to that in seedling leaves. 【Conclusion】Compared with the relative expression level of Pi9 gene at the seedling stage in IRBL9-W, the significant decrease of Pi9 gene expression level in panicle of IRBL9-W could be the cause of susceptibility to M. oryzae at late-booting stage.

Key words: Oryza sativa L., Magnaporthe oryzae, Pi9, seedling stage, adult-plant stage

摘要：

【目的】Pi9是一个广谱稻瘟病抗性基因,田间病圃监测发现持有Pi9的水稻单基因系IRBL9-W在苗期高抗稻瘟病,但却感穗瘟。探明水稻单基因系IRBL9-W在苗期抗病而孕穗末期感染穗颈瘟的原因,为Pi9基因在水稻抗病育种中的有效利用提供参考。【方法】利用从IRBL9-W穗颈瘟病斑上分离的8个单孢菌株以及实验室保存的单孢菌株Y363,在温室分别对单基因系IRBL9-W苗期和孕穗末期进行接种鉴定;并利用病原菌AvrPi9基因的特异引物对9个单孢菌株进行PCR扩增及产物测序;提取水稻单基因系IRBL9-W苗期叶片和抽穗期穗部的总RNA,通过半定量RT-PCR以及实时qRT-PCR分析Pi9基因在苗期和穗期的表达。【结果】在温室人工喷雾接种条件下,IRBL9-W在苗期对从穗颈瘟上分离的8个单孢及对照菌株Y363均表现为抗病;随机选取的2个从IRBL9-W穗颈瘟病样分离的单孢菌株(YX2-7-1和YX2-15-1)及对照菌株Y363对孕穗末期IRBL9-W注射接种,接种的植株表现出典型的穗颈瘟症状;AvrPi9的等位基因分析结果表明,与AvrPi9相比,Y363中的等位基因与AvrPi9完全相同,而从IRBL9-W穗瘟分离的8个单孢菌株中编码区与AvrPi9基因完全相同,但在编码起始位置上游-264 bp处缺失16 bp的一段序列。由于IRBL9-W苗期对这些菌株均表现抗病,推测这段序列的缺失并不影响AvrPi9基因的功能;实时qRT-PCR分析结果表明,Pi9基因在穗部的表达量为苗期叶片表达量的47.3%。【结论】在水稻单基因系IRBL9-W中,与苗期叶片中Pi9基因的表达量相比,Pi9基因在穗部表达量的明显降低可能是造成IRBL9-W穗期感稻瘟病的原因。

关键词: 水稻, 稻瘟病菌, Pi9, 苗期, 成株期

Shufang LIU, Liying DONG, Xundong LI, Wumin ZHOU, Qinzhong YANG. Different Reactions of Rice Monogenic Line IRBL9-W Harboring Pi9 Gene to Magnaporthe oryzae Containing AvrPi9 During Seedling and Adult-plant Stages[J]. Chinese Journal OF Rice Science, 2021, 35(3): 303-310.

刘树芳, 董丽英, 李迅东, 周伍民, 杨勤忠. 持有Pi9基因的水稻单基因系IRBL9-W对稻瘟病菌苗期和成株期抗性差异[J]. 中国水稻科学, 2021, 35(3): 303-310.

Figures/Tables 5

Table 1 Pathotyping of Magnaporthe oryzae strains on monogenic lines.

单基因系 Monogenic line	抗性基因 R gene	稻瘟病菌株 Magnaporthe oryzae strains
单基因系 Monogenic line	抗性基因 R gene	YX2-2-1	YX2-3-2	YX2-5-1	YX2-6-1	YX2-10-1	YX2-14-1	YX2-7-1	YX2-15-1	Y363
IRBLA-A	Pia	R	R	R	R	R	R	S	S	S
IRBLI-F5	Pii	R	S	R	S	S	R	S	S	S
IRBLKS-F5	Piks	S	S	S	S	S	S	S	S	S
KRBLK KA	Pik	S	S	S	S	S	S	S	S	S
KRBLKP-K60	Pikp	S	S	S	S	S	S	S	S	R
KRBLKH-K3	Pikh	R	R	R	R	R	R	R	S	R
IRBLZ5-CA	Pi2	S	S	S	S	S	S	S	S	S
IRBLZ FU	Piz	S	S	S	S	S	S	S	S	S
IRBLZT-T	Pizt	R	R	R	R	R	R	R	R	R
IRBLTA-K1	Pita	S	S	S	S	S	S	S	S	S
IRBLB-B	Pib	S	S	S	S	S	S	S	S	S
IRBLT-K59	Pit	R	R	R	R	R	R	S	S	S
IRBLSH-B	Pish	S	S	S	S	S	S	S	S	R
IRBL1-CL	Pi1	R	R	R	R	R	R	R	S	S
IRBL3-CP4	Pi3	S	S	S	S	S	S	S	S	S
IRBL5-M	Pi5	R	R	R	R	R	R	R	R	R
IRBL7-M	Pi7	S	S	S	S	R	S	S	S	R
IRBL12-M	Pi12	S	S	S	S	S	S	S	S	R
IRBL19-A	Pi19	S	S	S	S	R	S	S	S	S
IRBLKM-TS	Pikm	R	R	R	R	R	R	R	S	S
IRBL20-IR24	Pi20	R	R	R	R	R	R	R	R	R
IRBLTA2-PI	Pita2	S	S	S	S	S	S	R	S	R
IRBL11-ZH	Pi11	S	S	S	S	S	S	S	S	S
IRBL9-W	Pi9	R	R	R	R	R	R	R	R	R
LTH	-	S	S	S	S	S	S	S	S	S

Fig. 2. Amplification of AvrPi9 from different M. oryzae strains. M, DNA molecular marker; DL2000; Lane 1 to Lane 9 refer to YX2-2-1, YX2-3-2, YX2-5-1, YX2-6-1, YX2-10-1, YX2-14-1, YX2-7-1, YX2-15-1 and Y363, respectively.

Table 2 Investigating results of panicle blast of IRBL9-W inoculated with three M. oryzae strains at late-booting stage.

稻瘟病菌株 M. oryzae strain	发病级别 Disease scores
稻瘟病菌株 M. oryzae strain	0	1	3	5	7	9
YX2-7-1	0^a	0	0	1	1	13
YX2-15-1	0	0	0	0	1	13
Y363	0	0	0	0	1	14
空白对照CK^b	15	0	0	0	0	0

Fig. 1. Symptoms of monogenic line IRBL9-W inoculated with M. oryzae fungus at late-booting stage and seedling stage. A–C, Symptoms of monogenic line IRBL9-W inoculated with M. oryze strains YX2-7-1, YX2-15-1 and Y363 at late booting stage, respectively. D–F: Symptoms of monogenic line IRBL9-W (left) and Lijiangxintuanheigu (right) inoculated with M. oryzae strains YX2-7-1, YX2-15-1 and Y363 at seedling stage, respectively.

Fig. 3. Expression level of Pi9 gene. A, Expression analysis of Pi9 gene by semi-quantitative RT-PCR method. The cDNA prepared from panicles (P) of adult plants and seedling leaves (L) of IRBL9-W carrying Pi9 gene was used as template for PCR amplification, with Actin 1 as reference gene; B, Comparative analysis of relative expression level of Pi9 gene in panicle and seedling leaves of IRBL9-W by real-time quantitative RT-PCR.

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Different Reactions of Rice Monogenic Line IRBL9-W Harboring Pi9 Gene to Magnaporthe oryzae Containing AvrPi9 During Seedling and Adult-plant Stages

持有Pi9基因的水稻单基因系IRBL9-W对稻瘟病菌苗期和成株期抗性差异

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