Effector XopN of Xanthomonas oryzae pv. oryzae Plays a Virulent Role in Rice Varieties Harboring OsSWEET11 Homologs

doi:10.16819/j.1001-7216.2020.9108

Abstract

Abstract:

【Objective】 Xanthomonas outer proteins (Xops) are conservatively virulent effectors or virulence-assistant components secreted by the type Ⅲ secretion system in plant-pathogenic bacteria that falls into the Xanthomonas genus, including the rice bacterial blight pathogen. Xops were secreted to the outside of bacterial cells, and then transported to plant cells to play a pathological role. One of these proteins, XopN, secreted by the standard strain PXO99^A of the rice pathogen, has been implicated in the bacterial virulence by subverting plant immunity, but the specificity in the virulence role of XopN is unclear. 【Method】 By using the double crossover homologous-recombination technique, the XopN gene-knockout mutant ∆XopN and genetically complemented strain ∆XopN/XopN were generated under the background of the bacterial wild-type strain PXO99^A. By comparing both recombinant strains with PXO99^A in terms of bacterial multiplication in nutrition broth, the role of XopN in the bacterial multiplication was evaluated. Based on literatures, 14 varieties of rice were chosen for experimental assessments on the contribution of XopN to the spectrum of the bacterial virulence. By quantitative analyses of gene expression, the virulence of XopN was related to the effects of bacterial infection on foliar expression of the OsSWEET11 gene or its homologs in different rice varieties. 【Result】 The bacteria gain better multiplication in the medium with the presence of the XopN gene, in contrast to the gene-knockout mutation, which highly impairs the bacterial propagation level. Based on bacterial populations of PXO99^A, ∆XopN and ∆XopN/XopN multiplied in rice leaves, as well as severities of subsequently developed leaf blight symptoms, the 14 rice varieties fall into two categories. 1) Ten of the rice varieties (IRBB1, IRBB3, IRBB8, IRBB10, IRBB14, IR24, IRBB203, IRBB204, IRBB205 and IRBB211) display similar susceptibility to the bacteria no matter whether the XopN gene is present or not. 2)By contrast, the bacteria depend on XopN to cause strong virulence on rice varieties IRBB208, Asominori and Nipponbare. The bacteria also depend on XopN to perform a relatively low level of virulence on the rice variety IRBB13. In IRBB13 leaves, the virulence role of XopN is coincident with a marked suppression of the recessive resistance gene OsSWEET11/xa13 by the bacterial infection. On the contrary, the bacterial infection causes enhanced expression of the dominant disease-susceptibility gene OsSWEET11/Xa13 in leaves of Nipponbare and enhanced expression of the gene homologs in IRBB208 and Asominori leaves. In essence, the transcriptional promotion or inhibition of the OsSWEET11 homologs is provided by the bacterial strains PXO99^A and ∆XopN/XopN, instead of ∆XopN, suggesting the critical role of XopN in inducing the gene expression. In addition, XopN is also required for the bacteria to effectively induce hypersensitive response in leaves of the non-host plant tobacco. While a high extent of the hypersensitive response is induced by PXO99^A or ∆XopN/XopN strain, the response incurs a substantial reduction by ∆XopN. 【Conclusion】 XopN is a multifunctional effector and plays a virulence role in rice varieties that possess OsSWEET11 or its homologs. XopN is also necessary to the bacterial multiplication and has an additive contribution to the induction of hypersensitive response by the bacteria in a non-host plant.

Key words: Xanthomonas oryzae pv. oryzae, XopN, bacterial multiplication, virulence, OsSWEET1

摘要：

【目的】黄单胞菌细胞外泌蛋白质(Xanthomonas outer proteins, Xops)是植物病原黄单胞菌高度保守的毒性效应子或毒性辅助组分,通过细菌Ⅲ型分泌系统分泌到细菌细胞外部,然后转入植物细胞而发挥病理作用。水稻黄单胞菌水稻致病变种即水稻白叶枯病菌的标准菌株PXO99^A分泌的XopN是一种毒性效应子,通过影响寄主免疫反应而使水稻发病。但是,XopN对病菌毒性的影响是否因水稻品种的不同而异,还有待研究。【方法】利用同源双交换技术,先敲除了PXO99^A的XopN基因,获得了∆XopN突变体,又经过遗传互补,得到了回补菌株∆XopN/XopN。通过营养肉汤液体培养,测定了XopN对病菌繁殖能力的影响;根据文献选用14个水稻品种,通过接种实验,测定了PXO99^A对这些品种的毒性与XopN敲除或回补的影响;在XopN发挥毒性作用的水稻品种上,测定了隐性抗病基因OsSWEET11/xa13与显性感病基因OsSWEET11/Xa13受病菌侵染而表达的情况,分析了XopN敲除或回补的影响。【结果】在营养肉汤液体培养过程中,病菌突变体菌株∆XopN的繁殖速度明显低于野生型PXO99^A。PXO99^A、∆XopN和∆XopN/XopN接种水稻后,根据其在水稻叶片组织内的繁殖量及随后产生的白叶枯病症状的严重程度,将供试的14个水稻品种分为两种情况。一是感病程度与病菌XopN是否敲除或回补无关,这有10个水稻品种（IRBB1、IRBB3、IRBB8、IRBB10、IRBB14、IR24、IRBB203、IRBB204、IRBB205和IRBB211）,PXO99^A、∆XopN和∆XopN/XopN对它们的毒性无明显差别。二是XopN对病菌毒性发挥作用的水稻品种,包括高度感病的3个品种（IRBB208、Asominori和日本晴）和低感品种IRBB13。与PXO99^A或∆XopN/XopN相比,∆XopN对这4个水稻品种的毒性大为降低。在IRBB13上,病菌侵染对隐性抗病基因OsSWEET11/xa13在叶片内的表达发生抑制作用,这一效应与XopN的毒性功能相关。相反,日本晴显性感病基因OsSWEET11/Xa13却受病菌侵染的诱导,在叶片内的表达水平大幅度提高。IRBB208和Asominori携带OsSWEET11/Xa13同源基因,该同源基因在叶片内的表达水平也因病菌侵染而大幅提高。在这4个水稻品种上,PXO99^A和∆XopN/XopN能够诱导OsSWEET11/Xa13或其同源基因表达,但∆XopN无此作用。另外,XopN对病菌在非寄主植物烟草上诱发过敏反应有量变贡献,相比PXO99^A或∆XopN/XopN,∆XopN引起过敏反应的程度有所降低。【结论】XopN是一个有限广谱性效应子,在拥有OsSWEET11同源基因的水稻品种上发挥毒性作用。XopN也是病菌繁殖所需要的,对病菌在非寄主植物上诱导过敏反应有一定贡献。

关键词: 水稻白叶枯病菌, XopN, 细菌繁殖, 毒性, OsSWEET11

CLC Number:

Li LI, Xuyan MO, Tiantian LI, Liyuan ZHANG, Hansong DONG. Effector XopN of Xanthomonas oryzae pv. oryzae Plays a Virulent Role in Rice Varieties Harboring OsSWEET11 Homologs[J]. Chinese Journal OF Rice Science, 2020, 34(4): 368-382.

李丽, 莫旭艳, 李甜甜, 张丽媛, 董汉松. 白叶枯病菌效应子XopN在拥有OsSWEET11同源基因的水稻品种上发挥毒性作用[J]. 中国水稻科学, 2020, 34(4): 368-382.

Figures/Tables 13

Table 1 Xoo-relevant resistance gene and encoding proteins in the tested rice varieties.

水稻品种 Rice variety	抗病基因 Resistance gene	基因产物 Encoding protein	文献 Reference
IRBB1	Xa1	NLR (leucine-rich repeat receptor)	31-33
IRBB3	Xa3	LRR-RLK (leucine-rich repeat-receptor-like kinase)	31-33
IRBB8	xa8	Unknown	31, 34
IRBB10	Xa10	Executor	31, 33
IRBB13	xa13	OsSWEET11 (synonym Xa13)	31, 33
IRBB14	Xa14	Unknown	31
IRBB203	Xa3	LRR-RLK	31, 33
IRBB204	Xa4	RLK	31, 33
IRBB205	Xa5	TFIIA TF	31, 33
IRBB208	Xa8	Putative OsSWEET11	本研究This study
IRBB211	Xa11	RLK	AAY32487.1
IR24	Xa18	RLK	AAY32494
Asominori	Putative Xa13	Putative OsSWEET11	本研究This study
日本晴 Nipponbare	Xa13 (synonym Os8N3)	OsSWEET11 (synonym XA13)	33-36

Table 2 Bacterial strains and plasmid vectors used and created in this study.

菌株及质粒 Strains and plasmids	相关性状 The study-relevant characteristics	来源 Resource
菌株Bacterial strains 黄单胞菌Xanthomonas oryzae pv. oryzae
PXO99^A	PXO99的氮杂胞苷抗性克隆,也叫菲律宾小种6号 The azacytidine-resistant clone of PXO99, synonym Philippine race 6	本实验室This lab
∆XopN	PXO99^A XopN敲除突变体 PXO99^A XopN-knockout unmarked mutant	本研究This study
∆XopN/XopN	用质粒pHMXopN回补PXO99^A XopN敲除突变体 PXO99^A XopN mutant complemented with pHMXopN	本研究This study
∆XopQ	PXO99^A XopQ敲除突变体 PXO99^A XopQ-knockout unmarked mutant	本实验室This lab
质粒Plasmids
pMD19-Ts	基因克隆T载体,含Ap^r, Mob⁺, Mob(P), LacZa⁺, Amp^r Gene-cloning T-simple vector, Ap^r, Mob⁺, Mob(P), LacZa⁺, Amp^r	TaKaRa
pMD20-T	基因重组T载体,含Kan^r, Amp^r Gene-recombination T-simple vector, Kan^r, Amp^r	TaKaRa
pMD20-T::F1::Km::F2	重组的基因突变载体 Recombinant gene-mutagenicity vector	本研究This study
pHMI	多个pUC19重组的寄主更广的载体,含Sp^R Broad-host range vector with pUC19 polylinker, Sp^R	本实验室This lab
pHMI-XopN	XopN整合到pHMI载体lacz启动子上的重组基因表达载体 Recombinant gene-expression vector containing XopN fused to lacZ promoter of pHMI	本研究This study

Table 3 Information of primers used in PCR protocols for Xoo XopN knockout and complementation.

基因或基因片段 ^a Gene or partial sequence ^a	引物 Primer	酶切位点 Restriction site
F1	5＇-AAGCTTGCCGACATGAAGGTGTATGAGGTG-3＇	Hind Ⅲ
F1	5＇-ACTAGTTGCGGCACCCGATGCTGCTGC-3＇	SpeⅠ
F2	5＇-GGTACCTGGCCGCTGCCGGGTAATGC-3＇	KpnⅠ
F2	5＇-GAATTCGCGTGCTAATCAGGTTTGCCTTTG-3＇	EcoRⅠ
Kan	5＇-AGTGATTGCGCCTACCCGG-3＇	无None
Kan	5＇-ACGTCTTGAGCGATTGTGTAGG-3＇	无None
XopN	5＇-GACCATGATTACGCCAAGCTTCTGGCAACAGCATCCCTCC-3＇	Hind Ⅲ
XopN	5＇-GACCTGCAGGCATGCAAGCTTTTACGCCGGCGGCAGTGCCCGATC-3＇	Hind Ⅲ

Table 4 Information on rice genes and their primers used for RT-PCR and RT-qPCR analyses.

水稻品种 Rice variety	基因 Gene	登录号 Locus number	文献 Reference	引物 Primer	PCR产物长度 Product size / bp	用途 Subject
所有品种 All varieties	OsEF1α	AF030517	9-13	5＇-CGTGCCTGTGGGTCGTGTTG-3＇ 5＇-TCCTGGAGAGCCTCGTGGTG-3＇	120	通过RT-qPCR 进行基因表达量分析 Gene expression analysis by RT-qPCR
日本晴Nipponbare	Xa13 (OsSWEET11)	AK070510	10	5＇-TGGTTCTGCTACGGCCTCTT-3＇ 5＇-GGTACCAGAAGTAGAGCCCCATCT-3＇	103	通过RT-qPCR 进行基因表达量分析 Gene expression analysis by RT-qPCR
IRBB208, Asominori	假定的OsSWEET11同源物 Hypothetic OsSWEET11 homologs	AK070510	本研究 This study	5＇-TGGTTCTGCTACGGCCTCTT-3＇ 5＇-GGTACCAGAAGTAGAGCCCCATCT-3＇	103	通过RT-qPCR 进行基因表达量分析 Gene expression analysis by RT-qPCR
日本晴, IRBB208 Asomimori Nipponbare, IRBB208 and Asominori	SWEET11与其假定同源物 OsSWEET11 and its homologs	AK070510	本研究 This study	5＇-CCAAGGCCAAACCACACATG-3＇ 5＇-TAACTACTAATAATACCTG-3＇	1517 (Full length)	通过RT-PCR 进行基因克隆,验证产物序列 Gene cloning by RT-PCR and confir mation of the product sequences
IRBB13	xa13 (OsSWEET11)	DQ421394.1	33	5＇-GGCGTGCACAAGGTCGAGGT-3＇ 5＇-CTGGCCGTCATCGATCCGGC-3＇	205(3911-4116 in DQ421394.1)	通过RT-qPCR进行基因表达量分析 Gene expression analysis by RT-qPCR
				5＇-TACCCGAACGTGGGCGGCTT-3＇ 5＇-AGTAACTACTAATAATACCTGTC-3＇	752(3701-4453 in DQ421394.1)	通过RT-PCR进行基因克隆,验证产物序列Gene cloning by RT-PCR and confirmation of the product sequence

Fig. 1. XopN gene knockout from PXO99A. A, Verification of the gene-knockout recombinant vector pMD20-T::F1::Kan::F2 by PCR amplification of the indicated specific recombinant elements; M1, M2, DNA markers. B, Screening of the XopN-knockout mutant ∆XopN based on PCR amplification of Kan from genomic DNAs of the conjugate clones #1 to #4. M1, M2, DNA markers. C, Confirmation of the ∆XopN mutant by PCR amplification of the XopN from genomic DNAs of the conjugate clones #1 to #4 analyzed together with a control, which was the clone of bacteria transformed with the empty pMD20-T vector. CK, Blank control; M, DNA marker.

Fig. 2. Genetic complementation of the ∆XopN mutant. A, Escherichia coli colonial PCR analysis by using genomic DNA samples from the pHMI::XopN conjugate colonies #1 to #3; M, DNA marker. B, Restriction PCR analysis of the PCR products that were collected from A and digested by Hind Ⅲ. M1, M2, DNA markers. C, PCR analysis of genomic DNA samples from the pHMI::XopN conjugate colonies #1 to #3. CK, Control; M, DNA marker. In A to C, PCR was performed with the XopN-specific primers.

Fig. 3. Bacterial population of the indicated Xoo strains in NB medium. Data are shown as mean values ± SD. Significant differences exist between PXO99A and both mutants 16, 18, 20, 22 and 24 hours after inoculation based on ANOVA and Duncan’s test (n = 6, P < 0.05 or 0.01).

Fig. 4. Lesion Length of leaf blight caused by the Xoo strains in different rice varieties. Plants of various rice varieties at the bottom were inoculated by leaf-top clipping with bacterial suspensions of ∆XopN and PXO99A, respectively. Lesion length of leaf blight was measured two weeks later. Values are shown as mean ± standard deviation (SD) estimates of 3 independent experiments each involving 12 leaves of 6 plants. Asterisks indicate significant differences in the corresponding pair comparisons based on analysis of variance (ANOVA) and Fisher’s test at P < 0.05 (n = 12).

Fig. 5. Lesion length of leaf blight caused by the three Xoo strains in the two rice varieties. Plants of the rice varieties were inoculated by leaf-top clipping with bacterial suspensions of PXO99A, ∆XopN and ∆XopN/XopN, respectively. Lesion length of leaf blight was measured two weeks later. Data are mean values ± SD. Asterisks indicate significant differences in the multiple comparisons by ANOVA and Fisher’s test (P < 0.05, n = 12).

Fig. 6. Bacterial population of PXO99A, ∆XopN and ∆XopN/XopN in leaves of IR24 and IRBB208. Plants of rice varieties IR24 and IRBB208 were inoculated by leaf infiltration with bacterial suspensions prepared from each of the bacterial strains indicated at the bottom. Three days later, bacteria were recovered from leaves and scored as colony formation unit (cfu) in the given leaf size. Data are mean values±SD. Different letters indicate significant differences of multiple comparisons of data from different bacterial strains based on ANOVA and Duncan’s multiple-range test(P<0.01, n= 6).

Fig. 7. Populations of PXO99A, ∆XopN and ∆XopN/XopN bacteria multiplied in leaves of the 14 rice varieties. Plants of different rice varieties at the bottom were inoculated by leaf infiltration with bacterial suspensions prepared from each of the indicated bacterial strains on the top. Three days later, bacteria were recovered from leaves and scored as cfu in the given leaf size. Data shown are mean values ± SD. Different letters on the graph indicate that ANOVA and Duncan’s multiple-range test disclose significant differences of multiple comparisons of data from the corresponding rice varieties inoculated differently (P < 0.01, n = 3).

Fig. 8. Effects of XopN on expression levels of OsSWEET11 or its analogs in leaves of the 4 rice varieties. Plants were inoculated by leaf infiltration with bacterial suspensions prepared from each of the indicated bacterial strains. Immediately before inoculation (0 hour) and 24 hours after inoculation, inoculated leaves were excised and used to isolate RNAs. RNA samples were subjected to RT-qPCR in two combinations. One was IRBB13 RNA samples and a pair of primers specific to the recessive resistance gene OsSWEET11/xa13 of IRBB13. The other was RNA samples from additional 3 rice varieties and a pair of primers specific to the dominant disease-susceptibility gene OsSWEET11/Xa13 of Nipponbare. Relative levels of the genes were quantified as ratios of their transcript amounts to the transcript amount of OsEF1α, a constitutively expressed gene used as a reference. Data are mean values ± SD estimates. Different letters on graphs indicate significant differences of multiple comparisons of data from differently inoculated plants based on ANOVA and Duncan’s test (P < 0.05, n = 6).

Fig. 9. Effect of XopN on the ability of Xoo to induce hypersensitive response in tobacco leaves. A, A tobacco (Nicotiana benthamiana) leaf photographed at 36 h after infiltration with pure water (control) or with a bacterial suspension of every Xoo strain shown on the leaf. This photo represents 6 leaves of 3 plants that were treated similarly. B, Parts of leaves 24 h after infiltration with the indicated bacterial suspensions and stained with trypan blue to show micro-HR. Micro-HR was visualized with a microscopy and is present in the right image but absent in the left one. Both microscopic images represent 6 infiltrated leaves excised from 3 plants.

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