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. 2019 Jan 10;20(2):253.
doi: 10.3390/ijms20020253.

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics

Jinping Zhao  1   2 , Jingjing Xu  3   4 , Binghua Chen  5   6   7 , Weijun Cui  8   9 , Zhongjing Zhou  10 , Xijiao Song  11 , Zhuo Chen  12   13 , Hongying Zheng  14   15 , Lin Lin  16   17 , Jiejun Peng  18   19 , Yuwen Lu  20   21 , Zhiping Deng  22 , Jianping Chen  23   24 , Fei Yan  25   26
Affiliations

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics

Jinping Zhao et al. Int J Mol Sci. .

Abstract

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC⁻MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV⁻host interaction that might contribute to the symptom development.

Keywords: chloroplast proteomics; chloroplast targeting; nuclear/nucleus-encoded chloroplast-related protein; plant–virus interaction; rice stripe virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Influence of rice stripe virus (RSV) infection on N. benthamiana plants and the ultrastructure of chloroplasts from N. benthamiana leaves. (a) Phenotype of RSV-infected N. benthamiana plants at 25 dpi. (b) Viral RNA accumulation in mock plants and RSV-infected plants were analyzed by RT-PCR. (c) Ultrastructure of chloroplasts from RSV-infected N. benthamiana plants. Right panels are the magnified images of the line-boxed area in the left panels, respectively. MOCK, the mock-inoculated healthy N. benthamiana plants; RSV, the RSV-infected N. benthamiana plants.
Figure 2
Figure 2
Blue native PAGE of chloroplast membrane protein complexes isolated from an equal quantity of healthy leaves (MOCK) and RSV-infected leaves (RSV). MOCK, the mock-inoculated healthy N. benthamiana leaves; RSV, the RSV-infected N. benthamiana leaves. PSII-SC, PSII supercomplex; PSI-M, PSI monomer; PSII-D, PSII dimer; LHCII, PSII light-harvesting complex; LHCII-T, PSII light-harvesting complex trimer; LHCII-M, PSII light-harvesting complex monomer; Cytb6f, cytochrome b6/f complexes.
Figure 3
Figure 3
Analysis of the specificity of the chloroplast fraction by Western blot. MPRO, protein extracts of protoplasts from mock-inoculated healthy leaves; RPRO, protein extracts of protoplasts from RSV-infected leaves; MCHL, protein extracts of chloroplasts isolated from protoplasts from mock-inoculated healthy leaves; RCHL, protein extracts of chloroplasts isolated from protoplasts from RSV-infected leaves. UGPase, UDP-glucose pyrophosphorylase (cytoplasm marker); PC, plastocyanin (chloroplast marker); rbcL, RuBisCO large subunit.
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