Interaction between Rice stripe virus Disease-Specific Protein and Host PsbP Enhances Virus Symptoms  Kong Lingfang , Wu Jianxiang , Lu Lina , Xu Yi , Zhou Xueping   Molecular Plant  Volume 7, Issue 4, Pages 691-708 (April 2014) DOI: 10.1093/mp/sst158 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Effect of SP on PVX-Induced Symptoms. (A) Diagrams of PVX-based vectors expressing SP or SP mutants. Mutant muSP contains a stop codon at the 5’ end of the SP ORF. Mutant muSP1 lacks the N-terminus 33 amino acids (aa). Mutant muSP2 lacks the N-terminus 71 aa. Mutant muSP3 lacks 55 aa (aa 72–127) in the SP. Mutant muSP4 lacks the C-terminus 52 aa. Mutant muSPC52 only contains the SP C-terminus 52 aa. (B) Symptoms in the N. benthamiana plants infected with PVX vectors harboring SP, muSP, muSP1, muSP2, muSP3, muSP4, or muSPC52. Photographs were taken at 20 dpi. (C) Northern blot analysis of PVX RNA and RSV SP RNA in systemic leaves of the infected plants. The radioactive probes used in these assays are specific for the PVX CP or RSV SP. RNA loadings were estimated using ethidium bromide-stained agarose gels shown at the bottom of the panel. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Effect of RSV SP Expression on Rice and N. benthamiana Growth. (A) Images of 35S–SP transgenic rice (upper panel) and N. benthamiana (lower panel). An image of 35S–muSP4 N. benthamiana is also shown. (B) Northern blot analysis of SP or muSP4 mRNA accumulation in wild-type or transgenic plants using a radioactive probe specific for RSV SP. Two independent lines from each treatment were used. WT indicates that the total RNA was extracted from the empty vector-transformed plants. Ethidium bromide-stained agarose gels are used to show the RNA loadings (10 μg total RNA per lane). (C) Western blot analysis of SP accumulation in the 35S–SP transgenic rice and N. benthamiana. Two independent transgenic lines were used for each treatment. Equal amount of proteins extracted from various leaf samples were separated by SDS–PAGE and the SP accumulation was determined using an anti-SP antibody. M, protein molecular marker. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 The Presence of SP Enhances RSV-Induced Symptoms. (A) RSV-infected wild-type (WT) and 35S–SP transgenic rice plant at 20 dpi (upper panel) and the eighth to tenth systemic leaves from a representative infected plant (lower panel). (B) Time course of RSV symptom development in the WT and 35S–SP or 35S–muSP4 transgenic plants. Values represent the percentage of RSV-infected plants at various days post inoculation. Twenty plants were used for each treatment. (C) RSV-infected WT and 35S–SP or 35S–muSP4 transgenic N. benthamiana plants at 12 dpi. Representative leaves of the three plants are shown. (D) Northern blot analysis of RSV CP mRNA accumulation in the WT and 35S–SP or 35S–muSP4 transgenic plants infected with RSV. Samples were collected from the RSV-inoculated rice plants at 20 dpi and N. benthamiana plants at 12 dpi. For each sample, 10 μg total RNA was used. The membranes were probed with a radioactive probe specific for the RSV CP gene. Ethidium bromide-stained agarose gels were used to show RNA loadings in the study. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Assays of SP and PsbP Interaction. (A) SP and PsbP interaction detected in the yeast two-hybrid assay. Yeast strain Gold was co-transformed with the indicated plasmids shown on the left side of the panel and grown on a SD/-His/-Leu/-Trp medium containing Aureobasidin A (AbA) at three different concentrations. (B) GST pull-down analysis of interactions between SP and OsPsbP. GST–OsPsbP fusion protein or GST was incubated with equal amount of purified MBP–SP or MBP, and accreted to glutathione-sepharose beads. The beads were then washed and analyzed by Western blot assays using an anti-MBP antibody (upper) or an anti-GST antibody (lower). The two right membranes show the inputs of purified fusion proteins. The membranes were probed with an anti-MBP or an anti-GST antibody. (C) BiFC analysis of interactions between PsbP and SP in the agro-infiltrated N. benthamiana leaves. a, YFP fluorescence; b, bright field; c, overlaid fluorescence and bright field images. Scale bars = 50 μm. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Effect of PsbP Expression on Rice and N. benthamiana Growth. (A) Growth of the wild-type (WT), psbp mutant, and 35S–OsPsbP transgenic rice (upper panel), and growth of the WT, NbPsbP–RNAi, or 35S–NbPsbP transgenic N. benthamiana plants (lower panel). (B) qRT–PCR assay of PsbP gene expression in the WT, psbp mutant, or 35S–PsbP transgenic rice (gray bars), and the WT, NbPsbP–RNAi, or 35S–PsbP transgenic N. benthamiana (blue bars) plants. Relative accumulation level of PsbP transcript is normalized against the actin (for rice) or GAPDH (for N. benthamiana) gene. Error bar denotes the standard deviation of three biological replicates. (C) Northern blot analysis of PsbP mRNA in the WT, psbp mutant, or 35S–PsbP transgenic rice, and the WT, NbPsbP–RNAi, or 35S–NbPsbP transgenic N. benthamiana plants using a PsbP-specific radioactive probe. In each experiment, two independent lines were used. The bottom gel shows rRNA stained with ethidium bromide and is used to show loadings of each sample. (D) Western blot analysis of PsbP in the WT, psbp mutant, or 35S–PsbP transgenic rice and the WT, NbPsbP–RNAi, or 35S–NbPsbP transgenic N. benthamiana plant using an anti-PsbP antibody. Two independent lines were used for each treatment. Bottom panel shows the loadings of proteins. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Effect of PsbP Overexpression or Silencing on RSV Infection in Rice and N. benthamiana. (A) Wild-type, psbp mutant, and 35S–OsPsbP transgenic rice were inoculated with RSV and photographed at 20 dpi. Lower panel of (A) shows the eighth to tenth systemic leaves of the representative plants. (B) Wild-type, NbPsbP–RNAi, or 35S–NbPsbP transgenic N. benthamiana plants were inoculated with RSV and photographed 20 dpi. Leaves shown in the lower panel of (B) were from the above representative plants. (C) Time course of RSV symptom development in various rice (Os) and N. benthamiana (Nb) plants. Fifteen plants were used for each treatment in each experiment. (D) Northern blot analysis of RSV CP RNA accumulation in various plants at 20 dpi. The membranes were probed with a radioactive probe specific for RSV CP gene. The ethidium bromide-stained gels were used to show the RNA loadings. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Measurements of Photosynthetic Parameters. Assays of the net photosynthetic rate (Pn), electron transfer rate (ETR), and maximal quantum efficiency (Fv/Fm) of PSII were measured using the wild-type (WT) or transgenic N. benthamiana expressing SP, muSP4, or NbPsbP. The NbPsbP-silenced plant is labeled as NbPsbP–RNAi. Each treatment had 30 plants at the same developmental stage. * represents significant difference between treatments (Fisher’s LSD test, P < 0.05). Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 Electron Microscopy of Chloroplast in Various Leaf Tissues. Expression of RSV SP, knockout or knockdown of PsbP caused alterations in grana stacks. Thin sections were prepared from the fixed and embedded leaf tissues collected from the wild-type (WT), psbp mutant, 35S–SP transgenic, or RSV-infected rice. Similarly, sections were prepared the WT, 35S–SP, 35S–muSP4, or NbPsbP–RNAi transgenic, PVX-infected, or PVX–SP-infected N. benthamiana leaf tissues. After staining with uranyl acetate and lead citrate, the sections were examined under an electron microscope. Images shown in columns 2 and 4 are higher magnifications of the boxed areas in columns 1 and 3. White arrows indicate changes of stroma thylakoid membranes. Bars = 1 μm. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 9 Subcellular Localization of PsbP–GFP Fusion Proteins. Protoplasts were prepared from the wild-type or 35S–SP transgenic N. benthamiana leaves agro-infiltrated with pCHF3–GFP or pCHF3–PsbP–GFP at 36h post infiltration (dpi). The protoplasts were then examined under a confocal microscope. Panel a shows the localization of free GFP or PsbP–GFP fusion proteins in the WT and 35S–SP N. benthamiana protoplasts. Panel b shows red auto-fluorescence from chloroplast. Panel c is bright field images of the protoplast and Panel d is the overlaid images. Bars = 10 μm. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 10 Analysis of PsbP Accumulation in Chloroplast through Immunocytochemistry and Electron Microscopy. (A) Thin sections were prepared from the fixed and embedded leaf tissues harvested from the wild-type (WT), psbp mutant, 35S–SP transgenic, or RSV-infected rice and the WT, 35S–SP, 35S–muSP4, or NbPsbP–RNAi transgenic N. benthamiana leaf tissues. (B) Thin sections were prepared from PVX-infected or PVX–SP-infected N. benthamiana leaf tissues. All the sections were probed with an anti-PsbP followed by a Protein A-gold conjugate. Small images are higher magnifications of the boxed 0.25-μm2 areas. Chl, chloroplast; C, cytoplasm; V, vacuole; M, mitochondria. Bars = 0.2 μm. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 11 Western Blot Analysis of PsbP in Rice and N. benthamiana Chloroplast. Chloroplasts were isolated from leaves of the wild-type, 35S–SP transgenic, or RSV-infected rice and N. benthamiana leaves. Two representative plants were used for each treatment. The membrane was probed with an anti-PsbP antibody. Coomassie brilliant blue-stained Rubisco was used to show the loadings of individual samples. Molecular Plant 2014 7, 691-708DOI: (10.1093/mp/sst158) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions