Background
Thrip-transmitted tospoviruses (genus
Tospovirus, family
Bunyaviridae) cause significant loss in yield and quality of important vegetable, legume and ornamental crops in many parts of the world [
1]. Based on phylogenetic relationship of the nucleocapsid protein genes, tospoviruses had been categorized into two groups: the America group including
Tomato spotted wilt virus (TSWV) serogroup, and the Euro-Asia group including
Watermelon silver mottle virus (WSMoV) serogroup and
Iris yellow spot virus (IYSV) serogroup [
2]. With increased international trade of agricultural products, tospoviruses and their vector western flower thrips (
Frankliniella occidentalis) have spread across the Asian countries, infecting vegetables and horticultural crops. To date, nine members of
Tospovirus have infected economically important crops in Yunnan, Guizhou, Guangxi provinces in southwest China. The viruses identified in these regions are: the Calla lily chlorotic spot virus (CCSV) [
3], Capsicum chlorosis virus (CaCV) [
4], Groundnut yellow spot virus (GYSV) [
5], Hippeastrum chlorotic ringspot virus (HCRV) [
6],
Impatiens necrotic spot virus (INSV) [
7], Tomato necrotic ring spot virus (TNRSV) [
8], Tomato zonate spot virus (TZSV) [
9,
10], TSWV [
11] and WSMoV [
12]. Among these tospoviruses, TSWV and TZSV are the dominant species.
Most tospoviruses are 80–120 nm in diameter with tripartite RNA genomes, referred to as large (L), medium (M) and small (S) according to their molecular mass [
13]. The L RNA is negative-sense, and the M and S RNAs are ambisense. The L RNA encodes the RNA-dependent RNA polymerase (RdRp), and the M RNA encodes for the precursor of two glycoproteins (Gn and Gc) and a non-structural protein (NSm). The S RNA encodes the N protein and another non-structural protein (NSs).
TSWV has a worldwide distribution [
1], but TZSV was first reported in 2008 as a new tospovirus belonging to WSMoV serogroup in China [
9]. TSWV and TZSV share low amino acid sequence identities of the nucleocapsid protein (28.6 %), and other proteins (18.9–45.5 %) encoded by these two viruses. Despite the low sequence identities, both viruses have similar host ranges (tomato, pepper, potato, lettuce and weed species), and cause similar symptoms (ring spot, chlorosis and yellowing) on the same host plant species, making them difficult to diagnose.
The enveloped particles of tospoviruses are spherical or pleomorphic. Understanding the specific morphology of the virus is beneficial for rapid diagnosis using negative staining under electron microscope. The cytopathological features of tospoviruses infection were described mainly on two TSWV serogroup viruses, TSWV and INSV in the tobacco plant cells [
14,
15]. Ultrathin sectioning revealed that infected plant cells display characteristic structures such as doubly enveloped virions (DEV), singly enveloped virions (SEV), viroplasm (VP), nucleocapsid aggregates (NCA), paired parallel membranes (PPM), and amorphous inclusions (AI) [
15‐
17]. However, little is known of clustering and cellular distribution characteristics of virus particles caused by two distinct tospoviruses belonging to two different serogroups. In this study, we compared modified negatively staining, ultrathin section, immunogold labeling negative staining and high pressure freeze-electron microscopy to analyze the clustering and cellular distribution of virus particles of TSWV and TZSV infected host plants species including different tissues. We found major differences in virion clustering and cellular distribution features of TSWV and TZSV in tobacco, tomato and chilli.
Methods
Virus and plant sources
TSWV-KM (a tomato isolate of TSWV) and TZSV-441 (a tobacco isolate of TZSV) were used in this study. Both isolates were collected from diseased plants showing necrosis and ringspot symptoms in Kunming, Yunnan province, China in 2013. These viruses were mechanically inoculated onto leaves of the local host Chenopodium amaranticolor. Following two passages of single local lesions in C. amaranticolor, a single-lesion isolate of each virus was maintained on Nicotiana rustica. Virus isolates were propagated on N. tabacum cv. K326, N. tabacum cv Yunyan203, and N. Benthamiana and were confirmed the infections by ELISA.
Lettuce (Lactuca sativa), tobacco (N. tabacum cv Honghuadajinyuan, Yuanyan 87, K326), sweet pepper (Capsicum annuum) and tomato (Lycopersicon esculentum) plants infected naturally by TSWV and TZSV were collected from different areas of Yunnan province, China during 2011–2014.
Negative staining
Leaf or stem tissues freshly sampled from naturally infected or inoculated host plants were cut into thin slices, then the thin slices were submerged in 2 % (w/v) glutaraldehyde in 0.2 M PBS (pH7.2) at the room temperature. The carbon-coated grids were adsorbed in the sap for 5 min, dried using a wedge of filter paper, and stained with 1 % (w/v) ammonium molybdate in 0.2 M PBS (pH7.2) at the end of pH6.4 for approximately 2 min. Samples were observed under JEM 100CX-II transmission electron microscopy (JEOL Ltd, Tokyo, Japan).
Negative stain labeling were conducted by copper grid adsorption of the polyclonal antibody for 5 min, then the samples were mixed with 0.1 M PBS, and chopped with a blade, then copper grid was adsorbed in the sap for 5 min. The copper grid was placed in BL to block the antibody for 30 min, incubated in sheep-resistance against rabbit IgG gold (50x) for 45 min, rinsed with ddH2O three times, each time 10 min and stained with 2 % (w/v) ammonium molybdate solution for 3 min. Samples were observed under JEM100CX-II transmission electron microscopy (JEOL Ltd, Tokyo, Japan).
Ultrathin section
Tissues from diseased plants with conspicuous symptoms were cut into pieces of 1 mm × 1 mm × 2 mm and then were fixed using 2.5 % glutaraldehyde buffered with 0.2 M PBS (pH 7.2) for 24 h, rinsed in 0.1 M PBS and post-fixed for 2 h in 1 % osmium tetroxide. Fixed tissues were then dehydrated in a graded series of ethanol followed by propylene oxide for 1 h. The fixed samples were then embedded in Spurr’s medium. Thin sections of 60 to 70 nm were positive stained with 2 % (w/v) uranyl acetate for 15 min and then stained with 0.2 % (w/v) lead citrate for 15 min and samples were observed under HT7700 (Hitachi, Japan) transmission electron microscopy at 80 kV and photographed with a Gatan 830 CCD camera.
High pressure freeze -electron microscopy (HPF-EM)
The leaves with obvious symptoms were sampled and rapidly frozen in liquid nitrogen in a high pressure freezing (HPF) instrument (Leica EM HPM100, Germany). Following HPF, frozen samples were immersed in fixative for 7 days to undergo the freeze substitution (FS) (Leica EM AFS2, Germany). The procedure of the FS was as follows: the sample was immersed in acetone with 0.1 % tannic acid at −90 °C overnight and rinsed three times with pure acetone, then placed in fixative composed with 2 % osmium tetroxide in acetone at −90 °C for 72 h, −60 °C for 48 h and −30 °C for 48 h, respectively. The sample was rinsed three times in pure acetone at 0 °C for 20 min each, then placed in the Epon812 resin with 1:1 acetone: medium, 1:3 acetone: medium and pure medium. Finally, the sample was embedded in pure Epon812 resin using an embedding mold and heated at 60 °C for 48 h. The polymerized sample block was cut into ultra-thin sections of 90 nm thickness and observed in a transmission electron microscopy(Hitachi H-7650, Japan) after staining with 2 % uranyl acetate aqueous solution and lead citrate aqueous solution.
Conclusions
Our study uncovered TSWV and TZSV particles had the similar clustering characteristics forming singly particle (SP), doubly particles clustering (DPC), triple particles clustering (TPC), and multiple particles clustering (MPC). However, they have the distinct cellular distribution patterns in the different tissues and host plants. The results provide the basis for the future research on assembly, maturation and movement of virus particles in the cell of host plants.
Acknowledgments
This work was partially supported by grants from the 973 program (2010CB134501) of China, and Top Scientists Input Program (2013HA028), Open Project of Yunnan Key Lab of Agricultural and Biotechnology (2015YAB02) and Provincial Natural Science Foundation of Yunnan (2012CH007). We are grateful to Dr. Jenifer H. McBeath and Dr. Sijun Zheng for the revision of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Designed experiments and critically revised the manuscript: ZZ, XW. Performed experiments: KZ, JD, QF, ZZ, JH. Collected samples: JD, ZZ. Analyzed data: ZZ, KZ, JD. Drafted manuscript: ZZ, JD, XW. All authors read and approved the final manuscript.