MicroRNA-regulated Compatible Poplar <i>R2R3-MYB</i> Gene Expression Following the Infection of Virulent <i>Melampsora larici-populina</i>

LI Dan-lei; ZHANG Rui-zhi; WANG Feng; CHEN Qiao-li; NIU Chun-yang; LING Ya-ming; HAO Xin

doi:10.13275/j.cnki.lykxyj.2017.02.010

Volume 30 Issue 2

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MicroRNA-regulated Compatible Poplar R2R3-MYB Gene Expression Following the Infection of Virulent Melampsora larici-populina

College of Forestry, Northeast Forestry University, Harbin 150040, Heilongjiang, China

Corresponding author: WANG Feng, kingsummit@sina.com ;
Received Date: 2016-06-25

Abstract

Objective The poplar 'Robusta', susceptible to virulent rust E4 Melampsora larici-populina, provides suitable host material for the study of the plant host resistance/susceptibility to pathogen. Previously study indicates that the transcription factor (TF) plays an important role in determining the poplar susceptibility. In this study, the authors tried to determine the R2R3-MYB TF genes driving basal disease resistance and focus on microRNAs (miRNAs) regulation. Method Two small RNA (sRNA) libraries, a degradome cDNA library and 2 digital gene expression libraries were constructed for rust-inoculated (Rust+) and rust-free (Rust-) susceptible poplar 'Robusta' leaves through high-throughput sequencing. The expression of selected miRNA and target R2R3-MYBs were identified by RTq-PCR. Result Altogether, 230 R2R3-MYBs were identified. Among those R2R3-MYBs, the expression of 55 genes were suppressed/promoted following the drive straight of the rust, the others were unchanged. Based on the miRNA and degradome sequencing, 86 regulating interactions (22 miRNA to 18 R2R3-MYB) were identified. RTq-PCR of selected PC-3p-2521022_1 and PndMYB173 showed a negative post-transcriptional regulation. The R2R3-MYBs target genes predication indicated R2R3-MYBs related to many resistance and phytohormone pathways, such as response to salicylic acid stimulus, response to stress, response to jasmonic acid stimulus and response to abscisic acid stimulus. Conclusion The infection of virulent rust changed the expression of 23.9% R2R3-MYBs. The differential expression of R2R3-MYBs indicated that the rust fungi infection could not only switch on but also switch off R2R3-MYB TF genes. Under the stress of rust infection, the post-transcriptional regulation of R2R3-MYBs was regulated by the families of miR159 and miR858.
- microRNA,
- R2R3-MYB,
- Populus× euramericana,
- Melampsora larici-populina,
- compatible interaction

References

[1]	Bartel D P. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009, 136: 215-233. doi: 10.1016/j.cell.2009.01.002
[2]	Schwab R, Palatnik JF, Riester M, et al. Specific effects of microRNAs on the plant transcriptome[J]. Developmental Cell, 2005, 8: 517-527. doi: 10.1016/j.devcel.2005.01.018
[3]	Jover-Gil S, Candela H, Ponce MR. Plant microRNAs and development[J]. The International Journal of Developmental Biology, 2005, 49: 733-744. doi: 10.1387/ijdb.052015sj
[4]	Willmann M R, Poethig RS. Conservation and evolution of miRNA regulatory programs in plant development[J]. Current Opinion in Plant Biology, 2007, 10: 503-511. doi: 10.1016/j.pbi.2007.07.004
[5]	刘蕾, 杜海, 唐晓凤, 等. MYB转录因子在植物抗逆胁迫中的作用及其分子机理[J]. 遗传, 2008, 30(10): 1265-1271. doi: 10.3321/j.issn:0253-9772.2008.10.005
[6]	Li L. Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression[J]. Plant Journal, 2009, 58: 275-286. doi: 10.1111/j.1365-313X.2008.03778.x
[7]	Shim J S, Jung C, Lee S, et al. AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling[J]. Plant Journal, 2012, 73(3): 483-495.
[8]	Johnson E T, Dowd P F. Differentially enhanced insect resistance, at a cost, in Arabidopsis thaliana constitutively expressing a transcription factor of defensive metabolites[J]. J Agric Food Chem, 2004, 52: 5135-5138. doi: 10.1021/jf0308049
[9]	牛春阳, 王峰, 李丹蕾, 等. C14族R2R3-MYB基因调控杨树抗锈菌过敏性反应[J]. 北京林业大学学报, 2016, 38(7): 16-23.
[10]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant Journal, 2007, 49(4): 592-606. doi: 10.1111/j.1365-313X.2006.02980.x
[11]	Li Danlei, Feng Wang, Chao Wang, et al. MicroRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent Melampsora larici-populina[J]. Bmc Genomics, 2016, 17(1): 1-16.
[12]	Min C, Cao Z. Genome-wide expression profiling of microRNAs in poplar upon infection with the foliar rust fungus Melampsora larici-populina[J]. Bmc Genomics, 2015, 16(1): 173-184. doi: 10.1186/s12864-015-1362-2
[13]	陈俏丽, 李丹蕾, 王峰, 等. 欧美杂交杨Pnd-LRR3基因克隆及其抗锈菌侵染表达[J]. 东北林业大学学报, 2015, 3: 95-98.
[14]	By Yoav B, Daniel Y. The control of the false discovery rate in multiple testing under dependency[J]. Annals Statistics. 2001; 29: 1165-88.
[15]	Li Q, Lin Y C, Sun Y H, et al. Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa[J]. Proceedings of the National Academy of Sciences, 2012, 109(36): 14699-14704. doi: 10.1073/pnas.1212977109
[16]	Duan Y, Jiang Y, Ye S, et al. PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana[J]. Plant Cell Reports, 2015, 34(5): 831-841. doi: 10.1007/s00299-015-1745-5
[17]	Nawrath C, Metraux J P. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin. Plant Cell. 1999; 11: 1393-1404.
[18]	Navarro L, Dunoyer P, Jay F, et al. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science. 2006; 312: 436-439. doi: 10.1126/science.1126088
[19]	Liu P T, Wheelwright M, Teles R, et al. MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy. Nat Med. 2012; 18: 267-273. doi: 10.1038/nm.2584

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沈阳化工大学材料科学与工程学院沈阳 110142

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MicroRNAs(miRNA)是一类由20~22个核苷酸构成的非编码小RNA，可通过结合靶基因mRNA的3′非翻译区(3′-untranslated region，3′-UTR)使其降解或者阻碍其转录，从而在转录后水平控制基因表达^[1]。作为基因表达负调控子，miRNA主要在转录后水平调节植物基因表达^[2]，在植物新陈代谢、组织生长、器官发育分化以及细胞程序性凋亡中起重要作用^[3-4]。此外，miRNA在植物感受逆境胁迫而做出适应性调整的过程中发挥着重要的作用。特别是在病原侵染过程中，miRNA能够调控植物抗病途径中的关键靶基因来决定病原与寄主的亲和关系(compatible/incompatible interaction)。

MYB(v-myb avian myeloblastosis viral oncogene homolog)是一类在其N端含有MYB结构域的转录因子(transcription factor，TF)^[5]。MYB是植物中最大的一类转录因子家族，涉及水杨酸(salicylic acid，SA)、脱落酸(abscisic acid，ABA)及茉莉酸(jasmonic acid，JA)等植物抗病信号传导通路。拟南芥(Arabidopsis thalian)MYB30编码通过调控超长链(very-long-chain)脂肪酸的合成应答病原入侵^[6]。MYB44经由WRKY70激活SA介导的防卫反应，抑制JA介导的防卫反应，通过拮抗调节方式调控拟南芥对生物胁迫应答^[7]。水稻OsLTR1编码MYB转录因子调控JA防御反应^[8]。根据蛋白含有结构域的数量，MYB转录因子分为4种类型，其中R2R3-MYB转录因子参与植物响应生物和非生物胁迫等过程^[9]。目前发现的植物MYB在非生物胁迫表达过程主要受miR159、miR828、miR172和miR319等家族的miRNA调控。例如拟南芥ABA刺激促进miR159积累，miR159过表达则抑制MYB101和MYB33转录^[10]。

对落叶松-杨栅锈菌(Melampsora larici-populina)E4生理小种侵染欧美杨(Populus × euramericana)研究过程中^[11]，我们发现欧美杨抗病相关基因的表达并未发生预想的显著上调。以NBS-LRR基因为例，E4生理小种侵染后仅8个TIR-NBS-LRR表达上调，另有11个TIR-NBS-LRR和3个CC-NBS-LRR基因表达下调，其余21个NBS-LRR基因在E4生理小种侵染后表达量未发生显著变化。但，与抗病相关的NAC、WRKY和MYB等转录因子基因表达量却发生显著变化。大量研究表明miRNA参与植物生长发育和胁迫应答中的各个方面，可以在不同层面对靶基因进行调控，是基因表达精细调控必不可少的重要手段，处于复杂调控网络中的关键位置。关于木本植物miRNA在病原菌胁迫中的调控机制的研究相对较少，特别是“杨树-锈菌”体系的miRNA调控机制研究仍处于探索阶段^{[11, 12]}。为揭示欧美杨对E4锈菌感病的分子调控机理，本研究通过miRNA组、降解组(degradome)和基因表达谱(digital gene expression profiling，DGE)等高通量测序技术对锈菌侵染前后欧美杨R2R3-MYB基因及其调控miRNA进行研究。

1. 材料与方法

1.1. 试验材料与锈菌接种

锈菌接种组(Rust+)：E4强致病性落叶松-杨栅锈菌(东北林业大学森林病理学标本室保存)孢子悬浮液(ddH₂O，含0.004% Tween 20)接种欧美杨叶间隔期指数(leaf plastochrony index，LPI)为5~9的完全展开的叶片；对照组(Rust-)：ddH₂O(含0.004% Tween 20)处理欧美杨叶片^[13]，分别进行3个生物重复。接种量控制在0.01 mL·cm^-2 孢子悬浮液。孢子悬浮液对水琼脂培养基喷雾，显微镜下计数，计算接种密度ρ=60个孢子·cm^-2。Rust+和Rust-叶片同置于16℃，相对湿度60%，16 h光照·日^-1(80 uEm^-2 s^-1)培养。分别在时间点2 hpi(hours post inoculation)、6 hpi、12 hpi、24 hpi、48 hpi、96 hpi及168 hpi分别收集Rust+和Rust-叶片等量混合CTAB法提取各自处理组总RNA。

1.2. 组构建及测序

根据Illumina Small RNA制备试剂盒说明书(Illumina’s TruSeq Small RNA Sample Preparation Guide 1)构建Rust+和Rust- miRNA组。构建Rust+ 降解组cDNA文库。根据Illumina/Solexa标准步骤(directional mRNASeq sample preparation part #15018460 Rev. A, October 2010. Illumina, San Diego, CA, USA)构建Rust+和Rust- DGE文库。构建好的miRNA组、降解组cDNA文库和DGE文库送联川生物(LC Bio: http://www.lc-bio.com/)进行测序并应用llumina’s Sequencing Control Studio software version 2.8(SCS v2.8)、Illumina’s Real-Time Analysis version 1.8.70 (RTA v1.8.70)、Illumina’s Pipeline version 1.5和ACGT101-miR version 4.2(LC Sciences, Houston, TX, USA)等完成数据均一化^[14]和基础数据处理。

1.3. 生物信息学分析

筛选毛果杨(P. trichocarpa)和拟南芥基因组中的R2R3-MYBs，构建本地数据库，以DGE测序结果结合本实验室欧美杨转录组数据进行BLASTP比对(E值< 1e-10)筛选欧美杨R2R3-MYBs。根据DGE测序结果确定锈菌接种前后R2R3-MYBs表达量变化，log₂(rust+/rust-)≥1定义为表达量上调、log₂(rust+/rust-)≤-1定义为表达量下调、-1 < log₂(rust+/rust-) < 1定义为表达量未发生显著变化。根据伪发现率(false discovery rate，FDR)控制差异表达基因^[14]。根据miRNA组和降解组鉴定miRNA与其靶向R2R3-MYBs的调控关系。根据R2R3-MYBs结合域(binding motifs)筛选毛果杨基因组潜在的靶基因，并依据STRING(http://string-db.org/)收录的拟南芥同源基因预测R2R3-MYBs与靶基因的互做关系。对靶基因进行GO(gene ontology)富集分析(http://geneontology.org/)。

1.4. 荧光定量PCR

分别以7个时间点Rust+和Rust-第一链cDNA为模板，应用GoTaq 2-Step RT-qPCR System试剂盒在Bio-Rad iCycler iQ^TM5荧光定量PCR仪上进行扩增。根据测序结果合成PC-3p-2521022_1引物(mi-F：5′-GTG TCG TGC GGC ACT GAA-3′，mi-R：5′-GAT AAT TGG GCT CCG T-3′)和PndMYB173引物(MYB173-F：5′-GAA GAC GAG GCG TTA CAG AG-3′，MYB173-R：5′-GGC CCT GAT GAT TGT GTC AT-3′)。选用毛果杨18sRNA基因(18S-F：5′-CGA AGA CGA TCA GAT ACC GTC CTA-3′，18S-R：5′-TTT CTC ATA AGG TGC TGG CGG AGT-3′)作为内参基因(positive internal control)^[15-16]。使用2步法PCR进行40个循环并测定融解曲线。相对定量法计算3次重复试验初始模板量log₂(rust+/rust-)比值，两配对样本t检验差各时间点间异显著性。

3. 讨论

由miRNA介导的植物抗病性已有比较深入的研究^[17-19]。但miRNA转录后调控相关的植物感病性研究还处于起步阶段。以往研究表明效应因子诱导的感病性(effector-triggered susceptibility，ETS)决定杨树对锈菌的互作亲和关系(抗/感病性)。病原侵染后，植物基础抗病性(basal disease resistance)由病原诱导的免疫反应(pathogen-associated molecular patterns triggered immunity，PTI)，ETS和效应因子诱导免疫反应(effector-triggered immunity，ETI)的综合反应过程构成^[9]。PTI、ETS和ETI过程涉及到感病性增强因子1(enhanced disease susceptibility 1，EDS1)和非小种专化抗病因子1(non-race-specific disease resistance 1，NDR1)激活TIR-NBS-LRR和CC-NBS-LRR等抗性蛋白(R)完成抗病过程。在EDS1/NDR1到R蛋白的抗病性激活过程中涉及到复杂的信号传导和植物激素调节过程。在NDR1将细胞膜外的病原入侵信号传递到膜内后，一系列的TF即被激活，主要包括NAC、WRKY、AP2和MYB等。继而SA、JA和ET等植物激素相关的抗病路径启动。在这个复杂的寄主病原互作体系中，TF与其调控miRNA是决定寄主与病原亲和性的重要因素，如果抗病信号传导受阻或miRNA调控失活寄主即体现为亲和型症状，如果抗病信号顺利传导则寄主将产生HR和PCD等抗性症状。靶基因预测表明欧美杨差异表达R2R3-MYB可启动SA、JA、ET和脱落酸(abscisic acid)等多个植物激素路径。实际接种试验中欧美杨并未发生HR或PCD等症状，表明欧美杨的抗性信号传导过程可能出现了阻碍。本研究仅对一对R2R3-MYB转录后调控进行了荧光定量PCR验证，这还不足以检验出这种阻碍是否出现在R2R3-MYB转录后调控的层次上。因此，有必要对86对转录后调控关系逐一验证，综合分析欧美杨的感病性机理。

4. 结论

E4强致病锈菌侵染导致亲和型树种欧美杨23.9%的R2R3-MYBs表达量发生变化，锈菌侵染不但可以影响R2R3-MYBs的表达量，还可以启动或关闭R2R3-MYBs的表达。欧美杨在E4强致病锈菌胁迫条件下，R2R3-MYBs转录后主要受miR159和miR858家族的miRNA调控。已验证的R2R3-MYB与miRNA存在负向转录后调控关系。R2R3-MYBs靶基因涉及多个与免疫、植物激素相应和抗逆相关的生物过程。

Reference (19)

[1]	Bartel D P. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009, 136(): 215-233. doi: 10.1016/j.cell.2009.01.002
[2]	Schwab R, Palatnik JF, Riester M. Specific effects of microRNAs on the plant transcriptome[J]. Developmental Cell, 2005, 8(): 517-527. doi: 10.1016/j.devcel.2005.01.018
[3]	Jover-Gil S, Candela H, Ponce MR. Plant microRNAs and development[J]. The International Journal of Developmental Biology, 2005, 49(): 733-744. doi: 10.1387/ijdb.052015sj
[4]	Willmann M R, Poethig RS. Conservation and evolution of miRNA regulatory programs in plant development[J]. Current Opinion in Plant Biology, 2007, 10(): 503-511. doi: 10.1016/j.pbi.2007.07.004
[5]	刘蕾, 杜海, 唐晓凤. MYB转录因子在植物抗逆胁迫中的作用及其分子机理[J]. 遗传, 2008, 30(10): 1265-1271. doi: 10.3321/j.issn:0253-9772.2008.10.005
[6]	Li L. Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression[J]. Plant Journal, 2009, 58(): 275-286. doi: 10.1111/j.1365-313X.2008.03778.x
[7]	Shim J S, Jung C, Lee S. AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling[J]. Plant Journal, 2012, 73(3): 483-495.
[8]	Johnson E T, Dowd P F. Differentially enhanced insect resistance, at a cost, in Arabidopsis thaliana constitutively expressing a transcription factor of defensive metabolites[J]. J Agric Food Chem, 2004, 52(): 5135-5138. doi: 10.1021/jf0308049
[9]	牛春阳, 王峰, 李丹蕾. C14族R2R3-MYB基因调控杨树抗锈菌过敏性反应[J]. 北京林业大学学报, 2016, 38(7): 16-23.
[10]	Reyes J L, Chua N H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination[J]. Plant Journal, 2007, 49(4): 592-606. doi: 10.1111/j.1365-313X.2006.02980.x
[11]	Li Danlei, Feng Wang, Chao Wang. MicroRNA-mediated susceptible poplar gene expression regulation associated with the infection of virulent Melampsora larici-populina[J]. Bmc Genomics, 2016, 17(1): 1-16.
[12]	Min C, Cao Z. Genome-wide expression profiling of microRNAs in poplar upon infection with the foliar rust fungus Melampsora larici-populina[J]. Bmc Genomics, 2015, 16(1): 173-184. doi: 10.1186/s12864-015-1362-2
[13]	陈俏丽, 李丹蕾, 王峰. 欧美杂交杨Pnd-LRR3基因克隆及其抗锈菌侵染表达[J]. 东北林业大学学报, 2015, 3(): 95-98.
[14]	By Yoav B, Daniel Y. The control of the false discovery rate in multiple testing under dependency[J]. Annals Statistics, 2001, 29(): 1165-88.
[15]	Li Q, Lin Y C, Sun Y H. Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa[J]. Proceedings of the National Academy of Sciences, 2012, 109(36): 14699-14704. doi: 10.1073/pnas.1212977109
[16]	Duan Y, Jiang Y, Ye S. PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana[J]. Plant Cell Reports, 2015, 34(5): 831-841. doi: 10.1007/s00299-015-1745-5
[17]	Nawrath C, Metraux J P. Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin[J]. Plant Cell, 1999, 11(): 1393-1404.
[18]	Navarro L, Dunoyer P, Jay F. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling[J]. Science, 2006, 312(): 436-439. doi: 10.1126/science.1126088
[19]	Liu P T, Wheelwright M, Teles R. MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy[J]. Nat Med, 2012, 18(): 267-273. doi: 10.1038/nm.2584

序号 No.	GO术语 GO annotation	功能描述 Description	基因数 Gen number	p值 p value	伪发现率 FDR
1	GO: 0045087	先天免疫应答innate immune response	5	0.00035	0.0021
2	GO: 0002376	免疫系统进程immune system process	5	0.00046	0.0027
3	GO: 0006955	免疫应答immune response	5	0.00046	0.0027
4	GO: 0050896	响应刺激response to stimulus	34	1.40E-16	1.40E-15
5	GO: 0042221	响应化学刺激response to chemical stimulus	31	6.40E-22	7.90E-21
6	GO: 0010033	响应有机物response to organic substance	29	8.80E-25	1.20E-23
7	GO: 0009719	响应内源性刺激response to endogenous stimulus	28	4.30E-26	6.70E-25
8	GO: 0009725	响应激素刺激response to hormone stimulus	26	3.20E-24	4.30E-23
9	GO: 0005634	细胞核nucleus	21	1.50E-09	3.80E-08
10	GO: 0009751	响应水杨酸刺激response to salicylic acid stimulus	19	1.40E-27	2.20E-26
11	GO: 0006950	应激响应response to stress	19	7.90E-09	6.90E-08
12	GO: 0009753	响应茉莉酸刺激response to jasmonic acid stimulus	18	3.20E-25	4.60E-24
13	GO: 0009737	响应脱落酸刺激response to abscisic acid stimulus	18	4.90E-21	5.90E-20
14	GO: 0009628	响应非生物刺激response to abiotic stimulus	17	3.70E-10	3.30E-09
15	GO: 0006970	响应渗透压刺激response to osmotic stress	15	5.20E-16	4.80E-15
16	GO: 0009739	响应赤霉素刺激response to gibberellin stimulus	14	5.10E-20	5.70E-19
17	GO: 0009723	响应乙烯刺激response to ethylene stimulus	14	9.90E-19	1.10E-17
18	GO: 0010035	响应无机物response to inorganic substance	14	8.80E-17	8.90E-16
19	GO: 0009733	响应生长素刺激response to auxin stimulus	13	7.00E-14	6.30E-13

MicroRNA-regulated Compatible Poplar R2R3-MYB Gene Expression Following the Infection of Virulent Melampsora larici-populina