Volume 8, Issue 3, Pages 399-411 (March 2015) Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae  Chun-Lin Yang, Shan Liang, Hai-Yun Wang, Li-Bo Han, Fu-Xin Wang, Huan-Qing Cheng, Xiao-Min Wu, Zhan-Liang Qu, Jia-He Wu, Gui- Xian Xia  Molecular Plant  Volume 8, Issue 3, Pages 399-411 (March 2015) DOI: 10.1016/j.molp.2014.11.023 Copyright © 2015 The Author Terms and Conditions

Figure 1 Expression Profiles of the GhMLP28 Gene. (A) Expression pattern of the GhMLP28 gene in various organs of cotton plant. Root, stem, and leaf were sampled from the 14-day-old seedlings grown in a greenhouse, and flower was harvested from soil-grown plants on the day of anthesis. (B) Accumulation of GhMLP28 transcripts in cotton roots inoculated with V. dahliae. Total RNAs were extracted from roots of 14-day-old seedlings at 0–5 days after inoculation. (C) Expression of GhMLP28 after treatments with ET, JA, or SA. Total RNAs were extracted from roots of 14-day-old seedlings at 0–6 h after treatment, respectively. Error bars indicate the standard deviation (SD) of three technical replicates within one biological experiment. Three biological repeats were performed. Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 2 Increased Susceptibility of GhMLP28-Silenced Cotton Plants to V. dahliae. (A) Analysis of GhMLP28 expression levels. Total RNAs were extracted from leaves at 14 days post agroinfiltration and the expression level of GhMLP28 in VIGS plant was compared with that of control plant (transfected with TRV:00). (B) Disease symptom of GhMLP28-silenced plants infected by V. dahliae. (C) Rate of diseased plant (%) and disease index of the control and GhMLP28-silenced plants. Error bars in (C) indicate the SD (n = 80) of three biological replicates. Asterisks indicate statistically significant differences as determined by Student’s t-test (*P < 0.05). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 3 Enhanced Disease Tolerance of the Tobacco Plants Overexpressing GhMLP28. (A) Expression levels of GhMLP28 driven by the 35S promoter in transgenic tobacco lines (L1, L2, and L3). Histone 3 was used as an internal control. (B) Necrotic lesion area in leaf inoculated with V. dahliae in wild-type and GhMLP28-overexpressing (L2) tobacco plants. (C) Quantitative measurement of the necrotic lesion area shown in (B). (D) Symptoms of wild-type and GhMLP28 transgenic plants (L2) inoculated with P. parasitica for 10 days. (E) Rate of diseased plant (%) and disease index of wild-type and transgenic plants. Error bars indicate the SD of three biological replicates, n = 30 in (C) and n = 48 in (E). Asterisks indicate statistically significant differences as determined by Student’s t-test (*P < 0.05). WT, wild-type. Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 4 Interaction between GhMLP28 and GhERF6. (A) Yeast two-hybrid analysis of GhMLP28 and GhERF6 interaction. AD/T-BD/Lam and AD/T-BD/p53 represent the negative and positive control, respectively. β-Galactosidase activity was tested by X-gal filter assay. (B) Immunoblot assay of GhMLP28 and GhERF6 interaction. GST-tagged GhERF6 proteins were incubated with excess GhMLP28 proteins and the samples were subjected to native polyacrylamide gel separation. The bands were detected with anti-GST antibody. (C) LCI assay of the interaction between GhMLP28 and GhERF6. Luminescence imaging of N. benthamiana leaves was performed 48 h after co-infiltration with the same amount of Agrobacterium cells harboring constructs indicated on the left panel. (D) Quantification of relevant Luc activities in (C). Error bars represent the SD (n > 30) of three biological replicates. Asterisks indicate statistically significant differences as determined by Student’s t-test (**P < 0.01). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 5 DNA-Binding and Transcription Activation Activity of GhERF6. (A) EMSA analysis of the binding of GhERF6 to the GCC box. GhERF6 proteins were incubated with biotin-labeled probe (2× AGCCGCC) in the absence or presence of 2- to 8-fold of unlabeled probes for 30 min. (B) DLR assay of the transcription factor activity of GhERF6 in Arabidopsis protoplast. The empty vector pRT-BD and pRT-BD-VP16 were used as negative or positive control, respectively. Error bars represent the SD of three biological replicates with three technical repeats each. Asterisks indicate statistically significant differences as determined by Student’s t-test (*P < 0.05, **P < 0.01). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 6 Enhancement of GCC Box-Binding Activity of GhERF6 by GhMLP28. (A) EMSA analysis of the dose effect of GhMLP28 proteins on the binding activity of GhERF6 to the GCC box. GhERF6 proteins were incubated with a biotin-labeled probe (2× AGCCGCC) in the presence of His-tagged GhMLP28 proteins with indicated concentrations. (B) EMSA test for the presence of GhMLP28 in the up-shifted bands shown in (A). Anti-His antibody against His-tagged GhMLP28 was added in the reaction. The bands were detected by the method as in (A). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 7 Transient Expression Assay on GhMLP28-Enhanced Transcriptional Activation Activity of GhERF6. (A) Expression levels of GhERF6 and GhMLP28 in tobacco leaves transformed with indicated constructs in (B). (B) Luminescence signal on N. benthamiana leaves. Luminescence imaging was performed 48 h after co-infiltration with the same amount of Agrobacterium cells harboring constructs indicated on the left panel. (C) Luminescence intensity in N. benthamiana leaves measured by IndiGo software. Error bars in (C) represent the SD (n = 60) of three biological repeats. Asterisks indicate statistically significant differences as determined by Student’s t-test (*P < 0.05, **P < 0.01). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 8 Effect of GhMLP28/GhERF6 Silencing on PDF1.2 Promoter-Driven Luc Expression. (A) Transient expression assay of Luc in GhMLP28- or GhERF6-silenced cotton leaves. Top leaves (similar size) of GhERF6- or GhMLP28-silenced plants were injected with an equal amount of Agrobacterium cells harboring GhPDF1.2Pro:Luc or 35S:Luc. Luminescence imaging of cotton leaves was performed 48 h after infiltration with the constructs indicated on the left panel. (B) Luminescence intensity in cotton leaves measured by IndiGo software. Error bars indicate the SD (n = 60) of three biological repeats. Asterisks indicate statistically significant differences as determined by Student’s t-test (*P < 0.05, **P < 0.01). Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 9 Expression of PDF1.2 and PR5 Genes in GhMLP28-Overexpressing and GhMLP28-Silenced Plants. (A) Expression levels of GhPDF1.2 and GhPR5 in GhERF6- or GhMLP28-silenced cotton plants. (B) Expression levels of NtPDF1.2 and NtPR5 in GhMLP28 transgenic tobacco lines (L1, L2, and L3). Error bars indicate the SD of three technical replicates within one biological experiment. Three biological repeats were performed. Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions

Figure 10 Intracellular Distribution of GhMLP28 Proteins. (A) Subcellular localization of GhMLP28-GFP proteins in BY2 cells. The fluorescence of GFP-GhMLP28 was visualized under a fluorescent scope at 488 nm. n, nucleus. Bar, 25 μm. (B) Dependence of GhMLP28 nuclear distribution on the presence of GhERF6. Nuclear or cytoplasmic proteins were extracted from the roots of control (TRV:00) (lanes 1, 3, 5, and 7) or GhERF6-silenced cotton plants (lanes 2, 4, 6, and 8). Lanes 1, 2, 5, and 6, without inoculation; lanes 3, 4, 7, and 8, inoculated with V. dahliae; Histone 3, a marker of nuclear proteins; β-Actin, a marker of cytoplasmic proteins; CBB, Coomassie brilliant blue staining of nuclear and cytoplasmic proteins. Molecular Plant 2015 8, 399-411DOI: (10.1016/j.molp.2014.11.023) Copyright © 2015 The Author Terms and Conditions