Volume 9, Issue 9, Pages 1302-1314 (September 2016) Suppression of Jasmonic Acid-Mediated Defense by Viral-Inducible MicroRNA319 Facilitates Virus Infection in Rice  Chao Zhang, Zuomei Ding, Kangcheng Wu, Liang Yang, Yang Li, Zhen Yang, Shan Shi, Xiaojuan Liu, Shanshan Zhao, Zhirui Yang, Yu Wang, Luping Zheng, Juan Wei, Zhenguo Du, Aihong Zhang, Hongqin Miao, Yi Li, Zujian Wu, Jianguo Wu  Molecular Plant  Volume 9, Issue 9, Pages 1302-1314 (September 2016) DOI: 10.1016/j.molp.2016.06.014 Copyright © 2016 The Author Terms and Conditions

Figure 1 Disease Symptoms Induced by RRSV Infection in Rice. (A) Images of whole plants showing stuntedness and excess tillering. Scale bar, 15 cm. (B) Images of leaves with RRSV infection showing the phenotypes of curling, serration, and wider leaves. Scale bar, 5 cm. (C) Detection of the RRSV CP gene by RT–PCR. Rice actin 1 was used as an internal reference. (D) Detection of RRSV CP protein by western blotting. Tubulin was used as an internal reference. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 2 Effects of RRSV Infection on Rice miR319 and Its Targets. (A) Detection of miR319 accumulation in healthy (mock) and RRSV-infected (RRSV) rice plants by northern blot assays. (B) Measurement of miR319 accumulation in mock- and RRSV-infected rice plants by small RNA sequencing. (C) The relative expression levels of miR319 precursors, including miR319a and miR319b in mock- and RRSV-infected rice plants. (D) The relative expression levels of miR319-targeted genes in mock- and RRSV-infected rice plants. The expression levels were normalized using the signal from GAPDH. The average (±SD) values from three biological replicates of miRNA sequencing and qRT–PCR are shown. **p ≤ 0.01; *p ≤ 0.05. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 3 Increased Susceptibility of Rice to RRSV in Transgenic Lines with miR319 Overexpression. (A) Photos of mock-inoculated or RRSV-infected wild-type (Nipponbare, NPB) and miR319a/b-overexpressing (miR319a/b OE) transgenic lines. Scale bars, 15 cm (top) and 10 cm (bottom). (B) Detection of three RRSV genomic RNA segments in the indicated plants by northern blot assays. rRNAs served as loading controls. The RRSV RNA signals were quantified, and the relative levels are shown below the gels. (C) Detection of relative expression levels of RRSV CP in the RRSV-infected plants. Average values from three biological replicates are shown. Error bars represent SD. **p ≤ 0.01. (D) Morbidity of rice plants infected with RRSV. Data are shown as mean ± SEM (n = 3). Different letters at the top of each column indicate significant differences at p ≤ 0.05 (n = 3) by Student's t-test. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 4 Effect of TCP21 on RRSV Pathogenicity. (A) Schematic of the interaction of TCP21 with miR319 in wild-type (WT) and TCP21 Res plants, where bases that differ from those of WT plants are indicated in red. (B) The relative expression levels of TCP21 in mock (−) or RRSV-inoculated (+) WT and transgenic plants. The average values from three biological replicates of qRT–PCR are shown. Error bars represent SD. **p ≤ 0.01; *p ≤ 0.05. (C) Enhancement of RRSV pathogenesis in TCP21 IR transgenic lines. Scale bars, 15 cm (top) and 10 cm (bottom). (D) The relative expression levels of RRSV CP in the indicated plants with RRSV infection. The average values from three biological replicates are shown. Error bars represent SD. **p ≤ 0.01; *p ≤ 0.05. (E) Morbidity of rice plants infected with RRSV. Data are shown as mean ± SEM (n = 3). Different letters at the top of each column indicate significant differences at p ≤ 0.05 (n = 3) by Student's t-test. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 5 The JA Pathway Is Manipulated by miR319/TCP21 Activity. (A) qRT–PCR analysis of genes related to the JA pathway in indicated transgenic plants. (B) Endogenous JA levels in control groups, miR319 OE lines, TCP21 IR lines, and TCP21 Res lines. The JA concentration in leaves was measured by LC–MS, and three biological replicates were performed. Data are shown as mean ± SD. Error bars represent SD. **p ≤ 0.01; *p ≤ 0.05. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 6 Regulation of JA Pathway by RRSV Infection. (A) Microarray assays identified differentially expressed genes involved in JA synthesis and the JA signaling pathway. (B) The relative expression levels of genes related to JA synthesis and signaling in mock- and RRSV-infected rice plants. (C) JA levels in mock- and RRSV-infected rice plants were measured by LC–MS. Data are shown as mean ± SEM (n = 3). **p ≤ 0.01, significant difference between the WT and RRSV-infected rice plants by Student's t-test. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 7 MiR319 Is Possibly Involved in the Pathogenesis of Rice Black Streaked Dwarf Virus (RBSDV) in Wheat (Triticum aestivum). (A) Images of whole plants showing stuntedness, excess tillering, and leaves with curling, serration, and broadening. Scale bar, 15 cm. (B) Detection of the accumulation of mature miR319 in mock- and RBSDV-infected wheat plants by northern blot assays. (C) Relative expression levels of miR319 targets in mock- and RBSDV-infected wheat plants. The average values from three biological replicates of qRT–PCR are shown. **p ≤ 0.01; *p ≤ 0.05. (D) JA levels in mock- and RBSDV-infected wheat plants were measured by LC–MS. Data are shown as mean ± SEM (n = 3). **p ≤ 0.01, significant difference between WT and RBSDV-infected wheat plants by Student's t-test. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions

Figure 8 Scheme of the Role of miR319/TCP-Mediated Regulation of RRSV and RBSDV Infection in the WT Background. Solid lines represent regulatory links observed in rice, and dashed lines represent regulatory links observed in Arabidopsis and other plants. Arrows indicate positive regulation, and blunt-ended bars indicate inhibition. A line does not necessarily represent unique or direct regulation. Molecular Plant 2016 9, 1302-1314DOI: (10.1016/j.molp.2016.06.014) Copyright © 2016 The Author Terms and Conditions