Functional analysis

39 The geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top 40 disease in Iran and the newly established Becurtovirus genus type species. Although the biological features 41 of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, 42 and no information is available on the function of their viral genes. In this work, we demonstrate that 43 BCTIV V2, as the curtoviral V2, is also a strong local silencing suppressor in Nicotiana benthamiana and 44 can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the 45 silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, 46 being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector 47 also causes the induction of necrotic lesions in N. benthamiana like the ones produced during the HR, both 48 at local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant 49 showed that V2 is required for systemic infection but not for viral replication in a local infection. 50 Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 51 and plays a crucial role in viral pathogenicity and systemic movement. 52


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Geminiviruses are a group of insect-transmitted plant viruses that cause destructive diseases in major crops 60 worldwide (García-Arenal & Zerbini, 2019;Rojas et al., 2001). The virions of this family are twinned and 61 contain a single copy of circular single-stranded DNA, ranging in size from 2.5 to 3.0 kb. The

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family Geminiviridae includes more than 450 species organized into 14 genera according to the genome 63 organization (one-molecule genomes in monopartite and two-molecule genomes in bipartite) and other 64 biological features, such as host range and type of insect vector (Roumagnac et al., 2022(Roumagnac et al., ). 2012Zrachya et al., 2007), (ii) it is also required for viral movement and spreading of the virus through 82 the plant (Moshe et al., 2015;Padidam et al., 1996;Rojas et al., 2001;Rothenstein et al., 2007) and (

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This work aims to determine the role of BCTIV V2 in pathogenicity and its function as an RNA silencing 101 suppressor. This knowledge will help to study further the molecular mechanisms involved in the infection Generation of binary vectors: Gateway-compatible oligonucleotide primers BIV2Fw and BIV2RvSt or 120 BIV2RvNoSt were used to amplify BCTIV V2 full-length ORF, with or without stop codon, respectively, 121 and to generate entry clones by performing BP recombination reactions between the pDONR™ vector 122 (Gateway™ pDONR™/Zeo Vector) and the attB PCR products. Subsequently, these entry clones were 123 used for LR recombination reactions with Gateway™ destination binary vectors: pGWB2 yielding

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For BCTIV replication and infection analyses, plant DNA was extracted from the infiltrated (local) or the 158 apical (systemic) leaves of the infected plants at 6 or 28 days post-infiltration (dpi), respectively, digested 159 with DpnI to remove bacterial DNA in the infiltrated tissues (local infection) and then subjected to qPCR 160 analysis using primers BCTIV RTF and BCTIV RTR primers (Table S1), and N. benthamiana Elongation

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Expression of BCTIV V2 and BCTV V2 in agroinfiltrated tissues was determined by semiquantitative RT-163 PCR using the specific primers ClaI V2 F and SalI V2 (Table S1). EF1 α was used as an internal gene 164 control.

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For western blot analysis, 120 mg of leaf tissue per sample were used. Total protein was extracted by 2X

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3.1. V2 from BCTIV is a strong local PTGS suppressor 175 Amino acidic comparison among V2 from BCTIV with the homolog proteins from curtoviruses and 176 begomoviruses revealed that BCTIV V2 contains a conserved CK2/PKC (protein kinase CK2/protein 177 kinase C) phosphorylation motif (hereafter named P1) present in all V2 proteins and two putative CK2 and 178 PKC phosphorylation motifs predicted in BCTV V2 (named P2 and P3, respectively) are also present in 179 BCTIV V2, but not in begomoviral proteins (Fig.1B). Besides those phosphorylation motives, all V2 180 proteins have similar hydrophobic profiles displaying two hydrophobic regions at the N-terminus (hereafter 181 named H1 and H2), followed by a long hydrophilic region at the C-terminus (Fig. 1B). These hydrophobic 182 regions, along with P1, but not P2 or P3, have been proved to be essential for PTGS suppression activity 183 and pathogenicity in begomovirus and curtovirus (Chowda-Reddy et al., 2008;Luna et al., 2020). Besides 184 those conserved regions, the bioinformatic analysis identified a nuclear localization signal (NLS) present 185 in BCTIV V2 as well as in V2 from the curtovirus BCTV protein sequences while it was absent in most of 186 the begomoviral ones. (Supp. Fig. 1) (Dingwall & Laskey, 1991;Robbins et al., 1991).

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To test if BCTIV V2 functions as a PTGS suppressor, we carried out transient expression assays in N.

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benthamiana plants. Leaves were co-infiltrated with Agrobacterium cultures expressing GFP (35S:GFP) 189 and BCTIV V2 (thereafter named IVV2). As controls, leaves were co-infiltrated with 35S:GFP and the 7 empty vector (EV) (negative control) or a plasmid expressing the viral silencing suppressor V2 from BCTV fluorescence was still strong in the tissues co-infiltrated with 35:GFP and IVV2, as in leaves infiltrated with 193 BCV2. Conversely, in the patches infiltrated with the empty vector, fluorescence had disappeared almost 194 completely ( Fig. 2A). These differences in green fluorescence were confirmed by western blot analysis 195 using an anti GFP antibody. As expected, GFP protein accumulation in tissues infiltrated with IVV2 at 6 196 dpi was considerably higher than in tissues co-infiltrated with GFP and the empty vector. This enhancement 197 of GFP expression was as strong as the one produced by BCV2, proving that BCTIV V2 is also a strong 198 PTGS suppressor at a local level (Fig. 2B). Expression of viral genes in the infiltrated tissues was confirmed 199 by semiquantitative RT-PCR (Fig. 2C).  proteins, IVV2 and BCV2 can produce a slight delay in the silencing spreading, although none of them 213 seems to be blocking it completely. This delay was maintained until 30 dpi (Supp. Fig. 2, Fig. 3A).

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3.3. V2 from BCTIV cannot suppress short-range (cell-to-cell) spread of gene silencing 216 Agroinfiltrated 16c plants were also employed to study the effect of BCTIV V2 on the short-range 217 movement of the RNA silencing. For that, GFP expression was monitored with UV light in the cells 218 surrounding the infiltrated tissues at 6 dpi, to check if the silencing signal triggered by the GFP 219 overexpression had been able to exit from the infiltrated area and cause the appearance of a red ring around 220 the patch (Himber et al., 2003). In the plants co-infiltrated with 35S:GFP and the empty vector, the red ring 221 was observed at 6 dpi ( Fig. 3B). This red ring was also present around the patches infiltrated with BCV2, 222 but not in those infiltrated with P19, as previously described (Himber et al., 2003;Luna et al., 2017;Silhavy 223 et al., 2002). In the plants infiltrated with IVV2 a red ring was also visible at 6 dpi, showing that this viral 224 protein, as its Curtoviral counterpart, cannot block the cell-to-cell movement of the silencing signal to

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To achieve this, we generated a BCTIV infective clone containing a stop mutation in the eighth codon of  if V2 is required for viral replication, we performed a local infection assay. Total DNA was extracted at 6 272 dpi from the infiltrated leaves and viral DNA was quantified by qPCR. No differences in the amount of 273 viral-DNA accumulated in the infected tissues were detected in the leaves infected by wt BCTIV or BCTIV-274 V2stp (Fig. 6C). This result suggested that BCTIV V2 is required for viral movement but not for replication.