Viruses, a group of non-cellular organisms that rely on host machineries for replication, pose serious threats to human health and agriculture (Judson and Rabinowitz, 2021; Savary et al., 2019). In the last decades, begomoviruses (family Geminiviridae) transmitted by whiteflies of the Bemisia tabaci complex, emerge as plant pathogens of global significance (Fiallo-Olivé and Navas-Castillo, 2023). Begomoviruses are classified as monopartite and bipartite, which contain one and two circular single-strand DNA genome of 2.5-2.8 kb, respectively (Fiallo-Olivé et al., 2021). The genomes of bipartite viruses include DNA-A and DNA-B, with the former resembling the genome of monopartite begomoviruses (Rojas et al., 2005).
While in the Old World both monopartite and bipartite begomoviruses are widespread, the majority of characterized begomoviruses are bipartite and only a few monopartite begomoviruses have been reported in the New World (Souza et al., 2022). Intriguingly, bipartite begomoviruses from Old World and New World are divergent in several aspects. For example, of the six genes in DNA-A, AV2 can be found in Old World begomoviruses but not in New World viruses (Ha et al., 2008; Rojas et al., 2005). V2 proteins encoded in some monopartite begomoviruses and AV2 from a bipartite begomovirus induce plant hypersensitive response and systemic necrosis, respectively (Mubin et al., 2010; Roshan et al., 2018). Additionally, AV2 proteins encoded by some bipartite begomoviruses function as viral suppressors of RNA silencing (Chowda-Reddy et al., 2008; Roshan et al., 2018). Under this scenario, further dissection of the role of AV2 in Old World begomoviruses will promote our understanding of begomoviral pathogenesis, and in turn provides references for virus control.
Genetic variations often occur in viral genomes as an adaption to diverse stresses. Genetic changes in animal viruses are mostly directed by host immune systems (e.g., Sonneveld et al., 2012). Similarly, in response to antiviral machineries, plant virus may evolve rapidly through mutating the sequences that are targeted (e.g., Mehta et al., 2019). Additionally, chemicals that are administrated on diseased hosts for therapeutic purposes may induce directed genetic changes in viruses (e.g., zur Wiesch et al., 2011). Furthermore, for arthropod-borne viruses, arthropod vectors are essential for the completion of virus life cycle and may serve as selection pressure that directs the evolution of these viruses (e.g., Tsetsarkin et al., 2007). While how genetic variations promote virus adaptation to biotic factors has been revealed, whether and how they impact virus interaction with abiotic factors such as high temperature remain largely unexplored.
In a previous study, we characterized isolate Guangxi2017 of squash leaf curl China virus (SLCCNV), a bipartite begomovirus that were frequently found in South and Southeast Asia (Fig. 1A). Interestingly, in this isolate there is a naturally-occurring mutation in AV2. This mutation changes TGG to the stop codon TAG and thus may truncate AV2 proteins. The persistence of this mutant isolate in the field prompts us to explore the biological impact of this mutation. We first explored the impact of the naturally-occurring AV2 mutation on SLCCNV infectivity and transmission under ambient and high temperature. Since AV2 overlaps with other viral genes such as AV1, we then changed the start codon of AV2 to stop codon to analyze the role of AV2 in regulating SLCCNV infectivity under different temperatures. Our findings provide new insights into the regulation of begomoviral adaptation to high temperature.
Materials and Methods
Plants and insects
Four species of plants were used, namely zucchini (Cucurbita. pepo cv. Faguodongkui), squash (C. moschata cv. Mibennangua), cotton (Gossypium hirsutum cv. Zhe-Mian 1793) and transgenic GFP 16c Nicotina benthamiana (Gong et al., 2021). All plants were grown in insect-proof greenhouses under natural lighting at 25 ± 3°C. A culture of Asia II 1 whitefly of the B. tabaci complex (mtCOI GenBank accession code: DQ309077) was used as previous studies showed that Asia II 1 is an efficient vector of SLCCNV (Pan et al., 2020). Whiteflies were reared on cotton plants in insect-proof cages in artificial climate chambers at 26 ± 2°C, 60-80% relative humidity and 14/10 h light/dark cycles. In all experiments, female whiteflies of 0-4 days post emergence were used.
Mutagenesis and construction of infectious clones
All mutagenesis was performed based on SLCCNV isolate Guangxi2017 (GenBank accession codes: MG525551 [DNA-A] and MG525552 [DNA-B]). The codon TAG at 35 nt in AV2 was changed to TGG to generate DNA-A(AV2-WT), wherein AV2 was intact. The start codon of AV2 of SLCCNV Guangxi2017 DNA-A was change to stop codon to obtain DNA-A(AV2-null). The start codons of BC1 and BV1 of SLCCNV Guangxi2017 DNA-B were changed to stop codon to obtain DNA-B(BC1-null) and DNA-B(BV1-null). Briefly, SLCCNV DNA-A and DNA-B were amplified using primers containing BamHI restriction site, named SL-A-FLF and SL-A-FLR, SL-B-FLF and SL-B-FLR, respectively (Supplementary Table 1) and ligated into pClone007 Simple Vector (Tsingke Biotechnology, Beijing, China). To obtain the full-length DNA-A and DNA-B of mutant viruses, mutagenesis was performed using Fast Mutagenesis System (TransGen Biotech Co., Ltd., Beijing, China) with primers listed in Supplementary Table 1. The full-length genomes were then used to construct the corresponding infectious clones of DNA-A (pBINPLUS-1.3A) and DNA-B (pBINPLUS-1.9B).
Agrobacteria-mediated virus inoculation
Agrobacteria containing infectious clones of SLCCNV DNA-A or DNA-B were first cultured separately until OD600 reached 1.5-2.0, and then they were centrifuged and re-suspended in the infiltration buffer (10 mM MgCl2, 10 mM MES, 200 μM Acetosyringone). Re-suspended agrobacteria were incubated at room temperature for 1 h and then DNA-A was directly used for agro-inoculation or mixed with equal amount of DNA-B as specified in each experiment. One mL syringes were used to introduce the agrobacteria into both true leaves and cotyledons of zucchini or squash plants when they reached one or two true leaf stage. At 30 days post inoculation, the plants were subjected to symptom inspection or PCR detection of SLCCNV DNA-A.
PCR and quantitative PCR (qPCR) detection of SLCCNV DNA-A in plant and whitefly
For the detection of SLCCNV DNA-A in plants, the first apical fully-expanded leaves were harvested. One leaf from each plant was used as one sample and then subjected to DNA extraction using Plant Genomic DNA Kit (Tiangen, Beijing, China). For whiteflies, female adults that have fed on virus-infected plants for 96 h were collected as groups of 30 and then subjected to DNA extraction as described before (Shan et al., 2025). PCR detection of SLCCNV was performed with primers SL-A-PCRF and SL-A-PCRR (Supplementary Table 1). qPCR analysis of SLCCNV was performed using SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China) and CFX96 RealTime PCR Detection System (Bio-Rad, Hercules, CA, USA ) with the primers SL-AV1-RTF and SL-AV1-RTR for SLCCNV DNA-A, and primers WF-Actin-F and WF-Actin-R for whitefly actin, and Zu-Actin-F and Zu-Actin-R for zucchini and squash actin (Supplementary Table 1). The qPCR conditions were: initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec and extension at 60°C for 30 sec. Eight-twelve biological replicates (plants) and three technical replicates were conducted in the qPCR analysis. The relative level of virus quantity and gene transcription were normalized to plant or whitefly actin using 2−ΔCt method.
Acquisition and transmission of SLCCNV by whiteflies
Whiteflies were collected and released onto SLCCNV-infected plants for virus acquisition. Four days later, whiteflies were collected as groups of 30 for the analysis of virus quantity. Additionally, viruliferous whiteflies were collected as groups of 5 or 20 as designated in each experiment, and then released into leaf-clip cages enclosed on the leaves of zucchini or squash seedlings (one true-leaf stage). Four days later, whiteflies were removed and imidacloprid (20 mg/L) was sprayed, and the plants were kept in climate chambers for another month. All virus acquisition and transmission experiments were conducted in the climate chambers as mentioned above.
Comparison of infectivity among different genotypes of SLCCNV DNA-A under different temperatures
Two sets of comparison were conducted in this study, namely A(AV2-WT) vs. A(AV2-mutant) and A(AV2-WT) vs. A(AV2-null). Agrobacteria containing infectious clones of these DNA-As were cultured and re-suspended and then mixed with equal amount of DNA-B(WT) before being inoculated into zucchini or squash seedlings. Next, four temperature treatments (26, 34, 36 and 38°C) were conducted. Inoculated plants were placed in the climate chambers for ten days, and then moved to incubators (Panasonic, Osaka, Japan) at 26, 34, 36 or 38°C for another 20 days unless specified otherwise. The transfer of plants was conducted because we have only two climate chambers that were not sufficient for the high temperature treatments. The humidity and light/dark cycles of incubators were set as that in the climate chambers.
Analysis of virus quantity in plant sap and mechanical transmission efficiency
Plant sap was extracted following the protocol in King and Zeevaart (1974). Briefly, SLCCNV-infected zucchini plants were cut to obtain detached leaves (blade with petiole). Next, the basal part of the cut petiole was immersed in 20 mM ethylene diamine tetraacetic acid (EDTA) solutions, and kept in darkness in climate chambers for four hours. PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) was used to extract viral DNA from EDTA solutions. qPCR was then performed for these DNA samples, and the copy number was determined according to a standard curve that was made by qPCR reaction using serial dilution of pBINPLUS-SLCCNV-1.3A. Mechanical transmission was conducted as reported before with minor modifications (Lee et al., 2020). Briefly, apical symptomatic zucchini leaves were harvested and grounded in liquid nitrogen. Next, powders were dissolved in 0.01 M phosphate buffered saline (pH 7.4) (3:20, wt/vol), and the resultant mixtures were filtered using gauze to obtain crude virus extracts. The crude extracts were then inoculated onto zucchini leaves by rubbing with carborundum powder (600 mesh). All inoculated plants were kept in the climate chambers as mentioned above for one month, and then subjected to DNA extraction and detection of SLCCNV.
Analysis of functional redundancy between SLCCNV AV2 and BC1 or BV1
The agrobacteria containing various DNA-As (AV2-WT, AV2-mutant or AV2-null) and DNA-Bs (BC1-null or BV1-null) were cultured and re-suspended as described above. Next, six combinations were generated by mixing equal amount of DNA-As and DNA-Bs. Additionally, three treatments of DNA-A alone were conducted. As positive controls, the agrobacteria containing various DNA-As were mixed with DNA-B (WT), respectively. Zucchini plants were then inoculated and placed in the climate chambers. Thirty days later, inoculated plants were examined for symptom appearance and sampled for DNA extraction and PCR detection of DNA-A as described above.
Comparison of AV2 expression in SLCCNV-infected plants at 26, 36 or 38°C
The agrobacteria containing SLCCNV DNA-A(AV2-WT) and DNA-B(WT) were cultured, re-suspended and mixed as described above. Next, zucchini plants were inoculated and placed in climate chambers for 10 days, and then moved to incubators at 26, 36 or 38°C for 20 days, respectively. Plants were sampled for RNA extraction using TRIzol (Invitrogen) and then cDNA synthesis was conducted using Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biology). qPCR was performed using SLCCNV-AV2-RTF and SLCCNV-AV2-RTR for AV2, SLCCNV-AC1-RTF and SLCCNV-AC1-RTR for AC1, SL-A-RTF and SL-A-RTF for AV1, Zu-Actin-RTF and Zu-Actin-RTF for zucchini actin (Supplementary Table 1).
Virus competition assay
Virus competition assay was conducted for two combinations, namely DNA-A(AV2-WT) and DNA-A(AV2-mutant), and DNA-A(AV2-WT) and DNA-A(AV2-null). Briefly, zucchini plants were inoculated with equal amounts of two re-suspended infectious clones of DNA-A along with DNA-B(WT). Next, the plants were placed in the climate chambers for ten days and then moved to incubators at 26 or 36°C for 20 days. Next, plants were sampled for DNA extraction and virus detection. The percentage of each virus genotype in plants was determined as described before (Tsetsarkin et al., 2007). Briefly, genomic DNA was extracted and subjected to PCR using SL-A-EcoRI-F containing an EcoRI restriction site and SL-A-BamHI-R containing a BamHI restriction site. The PCR products were purified and ligated into pBINPLUS using EcoRI and BamHI. The recombinant plasmids were transformed into Escherichia coli strain DH5α, and 9-10 clones were sequenced for each sample. The sequencing results were aligned using DNAMAN 6.0 (Lynnon Biosoft, San Ramon, CA, USA), and the percentages of the two virus genotypes in each sample were manually calculated. Primers were listed in Supplementary Table 1.
Analysis of SLCCNV AV2-encoded protein as suppressors of post-transcriptional gene silencing (PTGS)
SLCCNV AV2 sequence was amplified and inserted into pCHF3 with XbaI and SalI to generate pCHF3-SLCCNV-AV2 with primers listed in Supplementary Table 1. This plasmid was then mobilized into agrobacteria strain EHA105. The pCHF3 empty vector, pCHF3-35S-GFP and pCHF3-p19 were used as mentioned before (Gong et al., 2021). Agro-inoculation and followed detection of GFP expression in 16c plants were conducted as described by Gong et al. (2021). Briefly, leaves of 16c plants were inoculated with four combinations of agrobacteria, namely pCHF3-35S-GFP + infiltration buffer, pCHF3-35S-GFP + pCHF3 empty vector, pCHF3-35S-GFP + pCHF3-p19, pCHF3-35S-GFP + pCHF3-SLCCNV AV2. Inoculated 16c plants were then kept in the climate chambers for the 26°C treatment or an incubator for the 36°C treatment. Four days later, GFP fluorescence was examined under a UV light and GFP protein level was determined by western blot using anti-GFP antibodies (Abmart, Shanghai, China).
Statistical analysis
For the comparison of virus infectivity, transmission efficiency and percentage of virus genotype, percentage data were arcsine square root transformed. qPCR data of quantity of virus were normalized to actin in plant or whitefly. qPCR data of SLCCNV AV1, AC1 and AV2 expression were normalized to zucchini actin. All comparisons were conducted using Student’s independent t test. To clearly illustrate the differences, the data of virus quantity in plants or whitefly in each of the experiments were normalized to that of control. All data were presented as the mean ± standard errors of mean (mean ± SEM) and differences were considered significant when P < 0.05. All statistical analyses were conducted using SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA) and Excel (Microsoft Corp., Redmond, WA, USA).
Results
Naturally-occurring mutation in SLCCNV AV2 and its effects on SLCCNV infectivity and transmission under ambient temperature
When examining Guangxi2017, an isolate of SLCCNV previously characterized by our laboratory, we found a mutation at 35 nucleotide (nt) in AV2 that changes the codon TGG to TAG (Fig. 1B). Interestingly, sequence analysis revealed that the protein encoded by non-mutated (wild type, WT) version of Guangxi2017 AV2 was identical to that of the AV2 of G25, another SLCCNV isolate previously characterized in this area (GenBank accession codes: AM260206 [DNA-A] and AM260208 [DNA-B]). Therefore, in this study, we re-named Guangxi2017 DNA-A as DNA-A(AV2-mutant). Based on DNA-A(AV2-mutant), we generated DNA-A(AV2-WT) (Fig. 1B).
To examine the impact of the mutation in AV2 ORF, we first compared the infectivity and transmission between DNA-A(AV2-WT) and DNA-A(AV2-mutant). Post agrobacteria-mediated virus inoculation, no significant difference in virus infectivity (percentage of symptomatic plants in all inoculated plants) and quantity of virus in zucchini plants was observed between DNA-A(AV2-WT) and DNA-A(AV2-mutant) in each of the three experiments (Fig. 2A, 2B). Similar results were obtained when squash plants were tested (Fig. 2C, 2D).
As begomoviruses are transmitted by whitefly under natural conditions (Fiallo-Olivé and Navas-Castillo, 2023), we then sought to determine whether AV2 mutation impacted virus acquisition and transmission by whitefly. When zucchini plants were used as the source of inoculum and test plants, no significant difference in the quantity of virus acquired by whitefly and virus transmission efficiency was found between DNA-A(AV2-WT) and DNA-A(AV2-mutant) (Fig. 2E, 2F). Similar results were obtained when squash plants were used as the source of inoculum and test plants (Fig. 2G, 2H).
Since AV2 mutation did not significantly affect SLCCNV infection in plants and transmission by whitefly vectors, we further analyzed other aspects of SLCCNV biology. We analyzed the virus quantity in plant sap and mechanical transmission efficiency as AV2 has been shown to impact the mechanical transmission efficiency in some bipartite begomoviruses (Lee et al., 2020). There was no significant difference in the quantity of virus in plant sap and mechanical transmission efficiency between DNA-A(AV2-WT) and DNA-A(AV2-mutant) (Fig. 2I, 2J). These findings suggest that the mutation at 35 nt in AV2 may not affect SLCCNV infection and transmission under ambient temperature.
Effects of AV2 start codon mutation on SLCCNV infectivity and transmission under ambient temperature
To further determine the role of AV2 in SLCCNV life cycle, we changed the start codon (ATG) of AV2 ORF to stop codon (TAG), and thereby generated DNA-A(AV2-null) (Fig. 1B). We then compared the infectivity and transmission between DNA-A(AV2-WT) and DNA-A(AV2-null). In three experiments conducted with zucchini plants, no significant difference was found in infectivity (percentage of symptomatic plants in all inoculated plants) between DNA- A(AV2-WT) and DNA-A(AV2-null) (Fig. 3A, 3B). Similar results were obtained when squash plants were tested (Fig. 3C, 3D). Further, there was no significant difference in the quantity of virus acquired by whitefly and mechanical transmission efficiency (Fig. 3E, 3F). These findings suggest that the change of start codon to stop codon in AV2 may not significantly modulate SLCCNV infection and transmission under ambient temperature.
SLCCNV AV2 could not functionally complement BC1 or BV1
AV2 of some begomoviruses has been shown to function as a movement protein, and for bipartite begomoviruses BC1 and BV1 encoded by DNA-B play a key role in virus movement (Gafni and Epel, 2002; Noueiry et al., 1994; Rojas et al., 2005). To explore the function of SLCCNV AV2, we examined whether AV2 may complement the function of BC1 or BV1. When SLCCNV DNA-As were inoculated alone into zucchini plants, none of the plants exhibited virus infection symptoms and viral DNAs could be detected only in few plants in two experiments (Table 1). When inoculated with DNA-B(BC1-null) or DNA-B(BV1-null), again these DNA-As did not induce any symptom and viral DNAs could be detected in only few plants (Table 1). As a positive control, in each experiment these DNA-As were inoculated with wild type DNA-B, and clear virus infection symptom including leaf curl and mosaic and DNA-A presence was determined in most of the inoculated plants. These results suggest that AV2 can not functionally complement BC1 and BV1 as both BC1 and BV1 are indispensable for SLCCNV infection whether AV2 is mutated or not.
Effects of AV2 start codon mutation on SLCCNV infectivity and transmission under high temperature
As host plants of SLCCNV, crops of Cucurbitaceae are mostly grown in spring and harvested in summer or autumn. Hence, high temperature becomes an inevitable environmental factor in the life history of SLCCNV. We thus sought to explore the impact of AV2 start codon mutations on SLCCNV biology under high temperature. At 26°C no significant difference in infectivity and quantity of virus in plants was found (Fig. 4A, 4B). At 34, 36 and 38°C, no significant difference was found in virus infectivity (Fig. 4A). However, at 34 and 36°C, the quantity of virus of DNA-A(AV2-WT) in plants was significantly higher than that of DNA-A(AV2-null) (Fig. 4B). When the plants treated with 36°C were presented to whiteflies, the quantity of virus acquired by whiteflies feeding on the plants infected by DNA-A(AV2-WT) was significantly higher than that feeding on DNA-A(AV2-null) infected plants (Fig. 4C). Subsequently, these viruliferous whiteflies were collected as groups of 5 and 20, and then used in virus transmission experiment. No significant difference in virus transmission efficiency between DNA-A(AV2-WT) and DNA-A(AV2-null) was found in two experiments (Fig. 4D, 4E). Virus competition assay showed that while at 26°C the percentage of DNA- A(AV2-WT) was close to 50% in zucchini plants infected simultaneously by the two virus genotypes, at 36°C the percentage of DNA-A(AV2-WT) increased to 100% (Fig. 4F).
We then compared the infectivity between A(AV2-WT) and A(AV2-mutant) at high temperature. The infectivity and virus quantity in plants did not differ between A(AV2-WT) and A(AV2-mutant) at either 34 or 36°C (Fig. 4G, 4H). Further, no significant difference in the percentage of A(AV2-WT) was found in mixed infected plants treated at 26 and 36°C (Fig. 4I). These findings suggest that the change of start codon to stop codon, but not mutation at 35 nt in AV2, decreases SLCCNV infection and transmission at high temperature.
Effects of high temperature on the expression of SLCCNV AV2
To explore the mechanisms underlying the modulation of SLCCNV infectivity by AV2 start codon mutation under high temperature but not under ambient temperature, we explored whether the expression of AV2 relative to other viral genes was induced at high temperature. We analyzed the expression of AV2 relative to other viral genes including AV1 and AC1 and zucchini actin in plants infected by DNA-A(AV2-WT). When compared to plants at 26°C, the expression of AV2, AV1 and AC1 relative to plant actin were all downregulated significantly at 36 or 38°C (Fig. 5A-5C, 5F-5H). However, no significant difference in the expression of AV2 relative to AV1 and AC1 was found in plants treated with different temperatures (Fig. 5D, 5E, 5I, 5J).
Function of SLCCNV AV2-encoded protein as suppressors of PTGS
Plant RNA silencing-mediated antiviral defense is activated at high temperature (Chellappan et al., 2005; Szittya et al., 2003), and AV2 of some bipartite begomoviruses has been shown to function as a viral suppressor of RNA silencing (Chowda-Reddy et al., 2008; Roshan et al., 2018). We thus speculate that SLCCNV AV2 may encode proteins that function as a viral suppressor of RNA silencing. We tested the function of AV2-encoded protein as a suppressor of PTGS at ambient (26°C) and high (36°C) temperature. At 26°C, the GFP fluorescence in 35S:GFP co-inoculated with SLCCNV AV2 was weaker than positive control (35S:GFP + P19), but much stronger than negative controls (35S:GFP + infiltration buffer and 35S:GFP + empty vector) (Fig. 6A). Similar results were obtained in western blot analysis of GFP level in leaf tissues (Fig. 6B). At 36°C, again SLCCNV AV2 increased GFP fluorescence and protein level (Fig. 6C, 6D). These results suggest that SLCCNV AV2-encoded protein functions as a suppressor of PTGS.
Discussion
In this study, we focus on two specific single nucleotide variations: a naturally-occurring mutation within AV2 ORF (TGG to TAG) and an introduced mutation that converts the start codon (ATG) to stop codon (TAG). The naturally-occurring mutation does not impact virus infection and transmission under ambient and high temperature, indicating that it is neutral. The introduced mutation, however, dampens virus infection at high temperature, indicating that AV2 ORF may play a role in the adaption of SLCCNV to high temperature.
Notably, the naturally-occurring mutation may truncate AV2 into a 11 amino acids (aa) peptide. There are several scenarios that might explain why this naturally-occurring mutation did not impact virus infectivity. The first is that the 11 aa peptide may function as the full-length AV2 in modulating SLCCNV infectivity. The second possibility that warrants experimental validation is that there are hidden ORFs inside AV2 ORF that are truly functional and are not affected by the mutation at position 35. Further investigations may explore these possibilities.
While the change of start codon to stop codon in AV2 does not seem to impact SLCCNV infectivity under ambient temperature, under high temperature this change significantly impairs SLCCNV infection. The change of start codon to stop codon may stop the transcription of the 11 aa peptide and thereby reducing SLCCNV infection. Alternatively, the presence of two stop codons may hinder the transcription of downstream ORFs in AV2. While further investigations are required to determine how the change of start codon to stop codon in AV2 modulate the transcription of viral genes, our findings clearly show that AV2 play a role in modulating SLCCNV infection under high temperature.
In the life cycle of begomoviruses, movements of viral genomic DNA and proteins play a significant role as viral genomes is replicated in nucleus and then transport to cytoplasm and adjacent cells (Breves et al., 2023). Here, we found that whether AV2 is mutated or not, both BV1 and BC1 are essential for SLCCNV infectivity, suggesting AV2 can not functionally complement BC1 and BV1. These findings indicate that AV2 function differ significantly from that of BC1 and BV1. Future investigations may carefully examine the function of these movement proteins, and thereby uncovering new determinants of the life cycle of bipartite begomoviruses.
Since mutation in AV2 ORF modulates SLCCNV infectivity under high temperature, we seek to explore whether the expression of AV2 is affected by high temperature and in turn facilitate SLCCNV infectivity. When calculated using plant actin as a reference, high temperature significantly decreased AV2 expression. The expression of AV1 and AC1 relative to plant actin were similarly downregulated by high temperature. Notably, AV2 expression level relative to AV1 and AC1 did not differ between ambient and high temperature treatments. These findings show that AV2 expression is downregulated to the same extent as the other viral genes, indicating that the augmentation of SLCCNV infectivity by AV2 under high temperature does not result from induced transcription of AV2. Another possibility is that the biological properties of AV2 protein are modulated by high temperature and thereby contribute to viral adaption to high temperature. For example, the activity of AV2 as movement protein and its stability may be altered under high temperature. In addition, the subcellular localization of AV2 may change with the increase of temperature. These factors may function alone or in combination, and should be examined in the future.
Another scenario is that AV2 promotes virus infectivity under high temperature by modulating other factors. For example, it is possible that high temperature directly modulates viral replication and movement machinery or host defense responses, and these modulations were altered by AV2, leading to AV2-induced increase in virus infectivity under high temperature. To explore this issue, future investigations may characterize the statues of viral replication and movement machinery or host defense responses such as the transcription of gene silencing-related gene in plants with or without AV2 upon high temperature treatment. Resultant findings will advance our understanding of the interplays among plants, begomoviruses and abiotic stresses.
RNA silencing is one of the major antiviral defense responses in plants that functions in a sequence-specific manner (Baulcombe, 2004). High temperature represents a major environmental factor that induces plant RNA silencing as manifested by increased amount of small interfering RNAs and expression of related genes (Fei et al., 2021; Szittya et al., 2003). Indeed, plant RNA silencing-mediated defenses against viruses such as begomoviruses have been shown to be stronger at high temperature (Chellappan et al., 2005). From the side of viruses, they encode viral suppressors of RNA silencing to sustain their persistence in host plants, and AV2 of some bipartite begomoviruses has been shown to function as a viral suppressor of RNA silencing (Chowda-Reddy et al., 2008; Roshan et al., 2018; Vance and Vaucheret, 2001). Here we validate the function of AV2-encoded protein as suppressors of PTGS under both ambient and high temperature, indicating that AV2 may promote SLCCNV infectivity under high temperature by encoding suppressors of PTGS. Further analysis may focus on the role of RNA silencing in the interplay between SLCCNV AV2 and plants under high temperature, thereby uncovering the mechanisms underlying AV2-promoted SLCCNV infectivity.
Taken together, here we identify a naturally-occurring mutation in SLCCNV AV2 and determine that this mutation did not impact SLCCNV infectivity and transmission under ambient and high temperatures. Moreover, we find that the change of the start codon of SLCCNV AV2 to stop codon significantly decreases virus infectivity under high temperature, but not under ambient temperature. Additionally, we explore the mode of action of SLCCNV AV2 and show that it may encode suppressors of PTGS. Our findings shed novel lights on the interactions between begomoviruses and abiotic factors that dictate viral pathogenesis.
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