Functional analysis of the nonstructural protein NSs of tomato zonate spot virus

Tomato zonate spot virus (TZSV), a member of the genus orthotospovirus, causes severe damage to vegetables and ornamental crops in southwest China. The NSs protein is an RNA silencing suppressor in various orthotospovirus like TZSV, but its mechanism and role in virus infection are poorly understood. Here, we observed that an NSs-GFP fusion protein was transiently expressed on the plasma membrane and Golgi bodies in Nicotiana benthamiana plants. The TZSV NSs gene was silenced and infiltrated into N. benthamiana and N. tabacum cv. K326. RT-qPCR and Indirect enzyme-linked immunosorbent assay (ID-ELISA) showed that the transcription and the protein expression of the NSs gene were inhibited by more than 90.00%, and the symptoms on silenced plants were alleviated. We also found that the expression of the Zingipain-2-like gene significantly decreased when the NSs gene was silenced, resulting in co-localization of the NSs-GFP and the Zingipain-2-like-mCherry fusion protein. The findings of this study provide new insights into the mechanism of silencing suppression by NSs, as well as its effect on systemic virus infection, and also support the theory of disease resistance breeding and control and prevention of TZSV in the field.

If the data are held or will be held in a public repository, include URLs, accession numbers or DOIs. If this information will only be available after acceptance, indicate this by ticking the box below. For example: All XXX files are available from the XXX database (accession number(s) XXX, XXX.).
• If the data are all contained within the manuscript and/or Supporting Information files, enter the following: All relevant data are within the manuscript and its Supporting Information files.
• If neither of these applies but you are able to provide details of access elsewhere, with or without limitations, please do so. For example: Data cannot be shared publicly because of [XXX]. Data are available from the XXX Institutional Data Access / Ethics Committee (contact via XXX) for researchers who meet the criteria for access to confidential data.
The data underlying the results presented in the study are available from (include the name of the third party • Introduction 28 Manucript without track changes Click here to access/download;Manuscript;Manucript -2021.11.14.docx Tomato zonate spot virus (TZSV), the dominant species, which belongs to the 29 orthotospovirus genus of the Bunyaviridae family in Yunnan Province, is transmitted 30 by thrips [1]. In recent years, TZSV has been prevalent in Yunnan, Guizhou, Guangxi, 31 and other regions of China and Southeast Asia. TZSV infection is often associated with 32 severe disease symptoms, including concentric rings and spots on fruits and necrosis of 33 leaves. TZSV has a very broad host range, infecting more than 20 types of economically 34 important crops and weed species, which belong to 7 families [2,3]. This prevalence 35 has not only led to production losses and quality problems for important vegetable and 36 ornamental plants in Southwest China but also seriously threatened economically 37 important crops for local farmers [4]. 38 TZSV consists of spherical, enveloped particles that become distributed in the 39 cytoplasm and the endoplasmic reticulum in mesophyll cells [1,5]. Like other members  PCR (RT-PCR) and the special primers (Table S1). The PCR fragments of NSs and Zingipain-2-like-mCherry, respectively. The Golgi marker ST52-mCherry was 134 amplified from the total RNA isolated from both tobacco and Arabidopsis [21].

136
The pTRV-pTV00-NSs constrcture was transformed into Agrobacterium tumefaciens  Confocal laser scanning microscopy and co-localization 155 The leaf epidermis was dissected from the areas of the agroinfiltrated N. 156 benthamiana leaves and placed in water between two coverslips. The confocal images 157 were captured with the inverted TCS SP8 and 10× water immersion objective lenses.

158
GFP was excited at a wavelength of 488 nm, and emissions were captured at 497-520  buffer was used as a blank control.

185
NSs was localized to the PM and Golgi bodies 186 Previous studies have shown that confocal laser scanning microscopy (CLSM) 187 was used to analyze the protein localized in living cells [22]. N. benthamiana is also a  be used as a calibration product to determine the copy numbers of the gene.

215
The PDS gene was used as a positive control to ensure the success of silencing. hypersensitive response [25,26]. To confirm whether the host factor for the Zingipain- The importance of NSs for tospoviral infection in plants was first discovered in 263 the early 1990s. A higher virulence of TSWV isolates and more severe symptoms were 264 observed with the elevated levels of NSs expression [27]. The TSWV NSs protein acts 265 as a suppressor of RNA silencing through binding small and long dsRNA and 266 suppresses short and long-distance viral accumulation and movement [28]. It also 267 represents the Avr factor of the Tsw resistance gene in pepper [29,30]. NSs of TSWV plants [37]. In this experiment, the temperature was strictly controlled, and thus, the 307 gene was silenced at relatively high levels, and the duration was relatively long.

Materials
The pTV00, pBINTRA, and pTV00-PDS vectors were provided by the Professor Jianqiang Wu's's laboratory, at the Kunming Institute of Botany, Chinese Academy of Sciences (KIB CAS) [20]. The vector of pCAMBIA-GFP, pBI121-mCherry were obtained from our lab in the institute of biotechnology and germplasm resources. The TZSV YN-Chili isolate was collected from the infected tomato field tomato plants in Yuanmou, Yunnan Province, China, and was maintained onin N. benthamiana [1]. N.  (Table S1).

TZSV inoculation
The frictionalfriction-based inoculation method was used to artificially infect N. tabacum cv. K326 and N. benthamiana plants at the 6 six-leaf stage. TZSV-infected N.
benthamiana leaves were homogenized in the PBS buffer (100 mg/mL) (containing 137 mM NaCl, 1 mM KH2PO4, 8 mMNa2HPO4·12H2O, and 3 mM KCl), and there afterafter, which the homogenate was applied uniformly to 3 leaves per plant (1 mL). Ten minutes after the TZSV inoculation, the inoculated plant leaves were rinsed with ddH2O.
PlantsPlant leaves inoculated with PBS buffer were used as controls, and five replicates were used for each sample. AfterFive days after inoculation for 5 days, when symptoms appeared, five replicates were used for each sample. The amplified products were subsequently analyzedimaged byusing a UVP gel-imaging system.

Plasmid constructs
(ⅰ) The fragments of the NSs gene to-be-silenced NSs gene were cloned in theinto pTV00 vectors. PCR was used to amplify the desired fragments with specific primers (Table S1)  (ⅱ) The sequences of NSs and Zingipain-2-like genes sequence was were amplified from the total RNA isolated from tobacco plants infected withby TZSV using reverse transcription-PCR (RT-PCR) and the special primers NSs-G-F/NSs-G-R (Table S1). The NSs-N and NSs-C PCR fFragments of NSs and Zingipain-2-like genes were digested with endonuclease Nco1 and Spe1 and inserted into vector of pCAMBIA-GFP and pBI121-mCherry using the same restriction sites to obtain pCAMBIA-NSs-N-GFP and pBI121-Zingipain-2-like-mCherrypCAMBIA-NSs-C-GFP, respectively. The Golgi markermarkers ST52-mCherry was amplified from the total RNA isolated from both tobacco and Arabidopsis [21].

Infiltration of the VIGs vector in Tobacco Leaves
The pTRV-pTV00-NSs vector constrcture was transformed into

Confocal laser scanning microscopy and co-localization assays
The leafLeaf epidermis waswere dissected from the areas of the agroinfiltrated leaf area of N.N. benthamiana leaves and placedmounted in water between two coverslips. The confocalConfocal images were captured with thean inverted TCS SP8 and 10× water immersion objective lenses. GFP was excited at a wavelength ofwith 488 nm, wavelength and emissions were captured at 497-520 nm captured. Moreover, mCherry waswere excited atwith a wavelength of 561 nm wavelength and emissions were captured at 585-615 nm captured. Images were processed using athe TCS SP8 and Adobe (San Jose, CA, USA) Photoshop.

ID-ELISA
The contentscontent of the genes proteins werewas tested by ID-ELISA according to the antibody instructions for antibodies to determine the antiviral activity of the VIGS. Leaves (0.2 g) were homogenized usingwith a mortar and pestle and diluted 1: 3 in a 1/3 dilution with PBS buffer. Crude extracts (100 µL) were added intoto ELISA plate wells and incubated at 37 °C for 2 h. The plate was then washed with PBST buffer.
TZSV NSs rabbit antibodies were diluted in a conjugationconjugate buffer. Afterward, and afterward, 100 µL of AP-conjugated goat anti-rabbit IgG-AP conjugate (Sigma, USA) was added to each well. The color-developing solution was dissolved in pnitrophenyl phosphate disodium hexahy drate (Sigma-Aldrich) in substrate buffer to obtain a final concentration of 1 mg/mL. The absorbance was determined at 405 nm was measure dusing athe ELx 808 microplate ELISA reader (Bio-Tek, USA). Healthy leaves were used as negative controls, PBS buffer was used as a blank control, and TZSV-infected leaves were used as positive controls. The PBS buffer was used as a blank control.

NSs wasis localized to the PM and Golgi bodies
Previous studies have shown that confocal laser scanning microscopy (CLSM) wasalways used to analyzeanalysis the protein localized in living cells [22]. N.N. benthamiana by agroinfiltration. We detected NSs-GFP was then detected to be associated within a structure that was the plasma membrane structures by CLSM (Fig   1 A-C). To determine whether the NSs-GFP bodies are colocalized with the Golgi stacks, we also checked the localization of NSs-GFP for Golgi bodies using the marker ST52-mCherry [23]. At 48 h afterAfter agroinfiltration onof N.N. benthamiana 48 h, we found that the NSs-GFP bodies are colocalized with the Golgi stacks (Fig 1 D-F), suggesting that the NSs-GFP protein was targetedtargets to the Golgi apparatus.

Silencing of the NSs gene
TRV vector wasis widely used to study the interactions between viruses and hosts and the functions of plant genes [24]. In the present study, Specificspecific primers (TZVNSsF1/TZVNSsR1) containing BamHI and HindIII restriction enzyme recognition sites were used to amplify the NSs gene fragments (Fig S1), and the DNA was inserted into athe pEASY-Blunt-Zero vector (TransGen, Beijing) for sequencing to ensure that the base sequences were not mutated.
The copies of the NSs gene were analyzedwas determined by RT-qPCR. The with the pTRV-pTV00-NSs vector before inoculation with TZSV (Fig 2A). Ssevere leaf shrinkage also occurred in positive control plants (inoculated only with only TZSV) exhibited severe shrinkage symptoms (Fig 2B)., and The shrinkage also appeared on the leaves of N. benthamiana plants injected with the pTRV-pTV00-NSs vector before inoculation with TZSV began to display light shrinkage symptoms (Fig 2A) ( Fig 3A, B). Taken together, theThe results indicateindicating that the pTRV-pTV00-NSs VIGS vector was successfullysuccessful constructed, and the NSs gene might be associated with TZSV infection.

Inhibition of the NSs protein expression
The NSs protein levelscontents waswere measured by ID-ELISA at 3 d, 5 d, 7d, and 9 ddays post-inoculation with TZSV,. and found to be significantly decreased The

The dependence of NSs gene silencing depending on the zingipain-2like gene
The Zingipain-2-like gene was a homocysteine protease, which possesses cysteinetype endopeptidase activity and participates in the regulation of plant-type hypersensitive response [25,26]. To confirm whether the host factor offor the  (Fig 4 A-C). Our results clarifiedclarify that the RNA silencing suppressor by (NSs) made closelyhad a close relationship with Zingipain-2-like when TZSV infected the plants.

Discussion
The importance of NSs for tospoviral infection in plants was first discovered in plants in the early 1990s., aA higher virulence of TSWV isolates and more severe symptoms werewas observed whenwith the elevated levels of NSs expression [27]. The TSWV NSs protein which acts as a suppressor of RNA silencing, through binding small and long dsRNA and suppressing suppresses local short and long-distance systemic silencing to enhanced viral us accumulation and movement [28]. It also represents the Avr factor of the Tsw resistance gene in pepper [29,30]. NSs of TSWV directly interacts with MYC2, a regulator of the JA signaling, to disable JA-mediated activation of terpene synthase genes and attenuates attenuate host defenses, increasingincreases the attractivenessattraction of the plants to thrips, toand thus transmittingtransmit the disease [31]. So far, the ability of NSs of TSWV NSs to counteract defense mechanisms mediated bythe RNA silencing defense mechanism in plants has been demonstrated was illuminated,; however, the mechanism of RNA silencing suppression by NSs in TZSV including microbe-associated (MAMP) or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is mediated by the corresponding membrane-anchored pattern recognition receptors (PRRs) inof plants, which serve as the first line of defense against the pathogen. Many plant viruses and their encoded proteins that could inhibit PTI were reported, such as like TSWV the NSs protein of TSWV that suppressed the production of reactive oxygen species (ROS), have been reported and PTI was inhibited [30,32,33]. In this study, we In this study, TRV vectors were used to construct VIGS vectors of the NSs gene tofor the analysis of their the functionsfunction. The results of RT-qPCR, andas well as the plant disease symptoms, showed that the gene replication was inhibited to up to 90%. ID-ELISA showed that the protein contents also significantly decreased. The high efficiency of gene silencing can be verifiedmeasured by sampling and testing immediately atwhen the onset of the disease appears on the plants. At the same time, temperature also hadhas effects on the silencing phenotypes inof plants [37]. In this experiment, the temperature was strictly controlled;, and thus, the gene was silenced at relatively high levels, and the duration was relatively long. TRV has a wide range of hosts, withand there is a significant difference in sensitivity to TRV between species and cultivars [38,39]. For instance, TRV sensitivity testing was carried out on 21 Gerbera gerbera cultivars, and the results revealed that only 5 cultivars showed photo bleached PDS-silencing symptoms on newly developed leaves [40]. In this study, the N. benthamiana leaves were bleached, but there were no phenotypic changes in the leaves of N. tabacum cv. K326, despite the fact that both species belongbelonging to the same genus, indicating that the TRV-VIGS vector exhibited differentdiffering sensitivities to different host species. TRV has a wide range of hosts, with a significant difference in sensitivity to TRV between species and cultivars [38,39]. For instance, TRV sensitivity testing was carried out on 21 gerbera cultivars, and the results revealed that only 5 cultivars showed photobleached PDS-silencing symptoms on newly developed leaves [40]. The VIGS method can be used for reverse genetics studiesresearch and the analysis ofto determine the functions of unknown genes.
In summary, our results presented here revealed that NSs, a suppressor of RNA silencing NSs ofin TZSV, was localized to the PM and Golgi bodies and might be alsoalso be associated with Zingipain-2-like to activate PTI-like responses taking advantage ofusing VIGS and subcellular localization predictionmethods. pTRV-pTV00-NSs +TZSV: means infiltrated pTRV-pTV00-NSs VIGS vectorconstruct and inoculated with TZSV; TZSV: positive control and means only inoculated with TZSV; pTV00-TZSV: means infiltrated pTRV-pTV00 VIGS vector and inoculated TZSV; CK: negative control and means the leaves with no treatment. All values are means ± SE. Means in a column followed by different letters are significantly different at P ≤ 0.05.

Fig 4 The NSs protein in TZSV NSs is co-localized with the Zingipain-2-like protein
NSs-G-F/ NSs-G-R (Table 1). The NSs-N and NSs-C PCR Fragments were digested with Nco1and Spe1 and inserted into pCAMBIA-GFP using the same restriction sites to obtain pCAMBIA-NSs-N-GFP and pCAMBIA-NSs-C-GFP, respectively. The Golgi markers ST52-mCherry was amplified from total RNA isolated from tobacco Arabidopsis [21]" has been changed into "The sequences of NSs and Zingipain-2-like genes were amplified from the total RNA isolated from tobacco plants infected with TZSV using reverse transcription-PCR (RT-PCR) and the special primers (Table S1).
The PCR fragments of NSs and Zingipain-2-like genes were digested with endonuclease and inserted into vector of pCAMBIA-GFP and pBI121-mCherry to obtain pCAMBIA-NSs -GFP and pBI121-Zingipain-2-like-mCherry, respectively. The Golgi marker ST52-mCherry was amplified from the total RNA isolated from both tobacco and Arabidopsis [21]." Answer: the sentence "The fragments of the to-be-silenced NSs gene were cloned in the pTV00 vectors." has been transferred to the location before the sentence "The vectors were then transformed into E. coli DH5α competent cells.".
Answer: The vector of pCAMBIA-GFP was obtained from our lab in the institute of biotechnology and germplasm resources, and insert this sentence before the sentence "The TZSV YN-Chili isolate was collected from infected field tomato plants in Yuanmou".
2) Vector and construct are different. pTRV-pTV00-NSs is a construct, not a vector.
Answer: Inoculation was performed using a 1-mL syringe, and the inoculation solution The reviewer asked where was pCAMBIA-GFP originally obtained from? Please also indicate where in the revised ms such information is provided.
Where is this information in the revised ms?
Where is the information in the revised ms? 2) Line 212-217: the sentence needs to be reorganized, Fig.2A goes before Fig. 2B Answer: Page 8, line 206-209: the sentence "TZSV was inoculated after 5 days, the positive control (inoculated with only TZSV) exhibited severe shrinkage symptoms ( Fig 2B), and the leaves of N. benthamiana plants injected with the pTRV-pTV00-NSs vector before inoculation with TZSV began to display light shrinkage symptoms (Fig   2A)," has been changed into "After 5 days of inoculation with TZSV, the shrinkage appeared on the leaves of N. benthamiana plants injected with the pTRV-pTV00-NSs vector before inoculation with TZSV (Fig 2A). Severe leaf shrinkage also occurred in positive control plants (inoculated only with TZSV) (Fig 2B);".