Plant Virus-Based Tools for Studying the Function of Gene and Noncoding RNA in Cucurbits

Abstract

Cucurbits are economically important crops worldwide. The genomic data of many cucurbits are now available. However, functional analyses of cucurbit genes and noncoding RNAs have been impeded because genetic transformation is difficult in many cucurbitaceous plants. The cucurbits-infecting plant viruses can be modified into useful tools for functional genomic studies in cucurbits, which provide alternative ways for rapid characterization of gene and noncoding RNA functions. This review will focus on the advancement and application of plant viruses-based gene silencing, gene expressing, and noncoding RNA regulation tools for studying the development, fruits, and stress of cucurbits. The features, advantages, and disadvantages of different plant virus vectors will be discussed in detail. We hope this review will provide guidance for studies on cucurbitaceous plants.

1. Introduction

Cucurbitaceae is one of the most genetically diverse plant families worldwide, which is viewed as the second-largest fruit and vegetable family after Solanaceae [1]. Biotechnological advances could empower the molecular characterization of functional genes in cucurbits and will inform new methods of breeding cucurbit species [2]. In recent years, intensive high-throughput genomic sequencing studies have made considerable advances in molecular phylogeny and genomics. The completion of genome sequencing of several cucurbitaceous crops increases the need for rapid gene function analysis tools [1, 3, 4].

Traditional transgenic approaches and CRISPR/Cas9-mediated genome editing technologies accelerated gene function characterization and crop improvement [5], while most economically important cucurbit crops are difficult to transform, or the transformation efficiency remains very low [2]. Since plant virus vectors could systemically infect host plants, fragments or the full length of the target genes of interest would be delivered into the whole plants to modulate gene expression within a short time by using simple operations [6]. Now, plant virus-mediated gene delivery systems have been successfully employed as alternative biotechnology tools for gene function studies, particularly in plant species recalcitrant for genetic transformation, including cucurbit plants [7]. Viruses can replicate in plant cells, and offer numerous advantages in gene overexpression, including their maximum levels of multiplication and concomitant levels of transient gene expression from viral genomes [8]. In the past years, several plant virus-based protein expression vectors have been widely used in gene overexpression to understand their functions. Furthermore, the discovery of posttranscriptional RNA silencing (PTGS) [9] and the development of modern sequencing tools spurred the development of virus-induced gene-silencing (VIGS) screens to knock down the specific host genes, which accelerated advances in investigating their functions [10, 11]. In addition, VIGS can also be used to study the roles of particular genes in metabolic pathways [12]. In plants, phytoene desaturase (PDS) is involved in the carotenoid biosynthesis pathway, and Knockdown of this gene results in albino phenotypes due to the absence of chlorophylls [13], making it widely used as an indicator for VIGS in plants.

MicroRNAs (miRNAs) are essential noncoding riboregulators of gene expression in plants, which influence the development and physiology of plants and responses to biotic and abiotic stresses [14]. Although much progress has been made in revealing miRNA functions in some model plans, their roles are still incompletely understood, especially in crop plants [15]. Compared with constitutive expression in transgenic plants, viral vectors have advantages in production and potentially rapidly. In the past years, plant viruses have been engineered into virus-based miRNA silencing (VbMS) vectors to inhibit the miRNA function in many plants, which are suitable for high throughput of analyzing miRNA function [16, 17, 18].

Recently, several plant viruses have been harnessed for CRISPR/Cas9-based genome editing of plants, which could both express Cas9 protein and deliver single-guide RNA (sgRNA) for genome editing [19, 20, 21, 22]. This type of virus-based strategy for gene editing is termed virus-induced genome editing (VIGE). Compared with traditional transgenic approaches, the VIGE process is more efficient and faster because the virus replication would give rise to high yields of sgRNAs and Cas proteins [23, 24].

Above all, the development of plant virus-based vectors has great implications for plant functional genomic studies. Different strategies are required for constructing virus vector-based systems due to their expression strategies and biological limitations [12], which lead to different suitable scenarios for the application of virus vectors. This review will discuss several plant virus-based tools used in cucurbits, including their construction strategies and examples of their applications.

2. Tobacco ringspot virus

Tobacco ringspot virus (TRSV) is the most well-characterized species of the genus Nepovirus in the family Comoviridae, which consists of a bipartite positive-strand RNA genome. Each RNA encodes a large polyprotein, which is proteolytically processed into mature proteins that encode different functions [25]. RNA1 is usually 8.1 to 8.4 kilobase (kb) nucleotides (nt) in length and encodes replication-related proteins, while RNA2 with a length from 3.4 to 7.2 kb nt usually encodes the capsid (CP) and movement protein (MP). TRSV can infect a wide range of plants, including cucurbits.

Zhao and co-authors developed TRSV virus-based vectors for foreign gene expression and VIGS in 2016. In the past few years, the application of viral 2A peptide allows the co-expression of multiple proteins from a single open reading frame (ORF), which prevents homologous recombination due to duplicated sequences for the protease cleavage sites, improving the stability of the vector [26]. In this research, they developed a vector with the 2A sequence upstream of the CP coding region to express green fluorescence protein (GFP). The 2A peptide could cleave the GFP protein from the polyprotein to produce partially free GFP. In addition, TRSV-based vectors showed the recovery phenotype, so GFP expression would decrease in the late infestation period [27]. To overcome this drawback, Fang et al. engineered TRSV vectors co-expressed GFP and heterologous viral suppressors of RNA silencing (VSRs) separated through two different 2A peptides in tandem, which produced stronger and more stable GFP expression in plants [16].

However, symptom recovery could be an advantage in the VIGS system for gene silencing experiments as viral symptoms usually confuse the silencing phenotypes. To develop the TRSV-based VIGS vector, researchers created a cloning site downstream of the CP stop codon for inserting the silencing gene sequence. This TRSV-based VIGS system caused clear silencing phenotypes and long duration of VIGS phenotype in cucurbits [16, 27]. Since TRSV-based VIGS vector was developed, it has been increasingly used in cucurbit crop studies such as the molecular mechanisms of defense-related genes and the multicellular trichome development due to its excellent silencing efficiency [28, 29].

In 2021, Fang et al. further developed the TRSV vector used for studying the function of miRNA in cucurbits. The phenotypes induced by TRSV-based miRNA silencing vector are obvious, and the efficiency of most TRSV-based miRNA silencing is high in both model plants and cucurbit plants. The silencing efficiency of miRNA was about 75.0–87.6% in loofah (Luffa aegyptiaca), and 68.8–75.0% in melon(Cucumis melo), which provides potential solutions for the functional study of cucurbit miRNAs [16]. It is noteworthy that this is the first report for the VbMS vector for the miRNA silencing in cucurbit plants.

3. Tobamoviruses

The genus Tobamovirus belongs to the family Virgaviridae, tobamoviruses assemble rod-shaped particles that are approximately 300–310 nm long and 18 nm wide and encapsidate a single-stranded, positive-sense RNA of 6.3–6.6 kb nt in length as its genome. Tobamoviruses can easily yield a large number of viral particles within a short time after they infect host plants [30], so these viruses can be used as good tools for the overexpression of genes [31]. Tobacco mosaic virus (TMV), the most well-characterized member of the genus Tobamovirus, has been extensively used for assessing gene function [32]. Tobamoviruses encode a 130-kDa protein (small replicase subunit), a 180-kDa readthrough protein (RNA-dependent RNA polymerase), an MP, and a CP. MP and CP were expressed via subgenomic RNAs (sgRNA) during infections [33]. Researchers showed that the most highly expressed viral gene is controlled by the CP subgenomic promoter (SGP) [34]. To establish a virus-based expression system, the foreign genes could replace CP. However, the recombinant viruses with genes only multiply in inoculated leaves [35]. It could be a good choice to place the heterologous gene at the terminal end of CP to express as a fusion coat protein if the protein expressed is small [36, 37]. The foreign genes could also be inserted in frame between MP and CP sequences, and the SGP in the CP gene was used to drive foreign gene expression. In the meanwhile, to prevent homologous recombination, CP SGP of other tobamoviruses was usually used to drive CP expression [38]. It is noteworthy that the length of the SGP should be empirically studied, as SGP length could affect virus accumulation and gene expression levels in plants [39]. TMV vectors designed with a duplication of the TMV 3′-UTR between the foreign ORF and the CP gene could increase the expression of the foreign gene [40].

3.1 Cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV), which mainly infects cucurbits under natural conditions, is a member of the genus Tobamovirus, CGMMV has been extensively exploited as suitable gene silencing and overexpression vectors in cucurbits [39]. In the early period, CGMMV was used to express the small protein in muskmelon. The expression of dengue virus type 2 envelope (E) protein and hepatitis B surface antigen (HBsAg) was achieved by inserting into the multiple cloning sites to cover the stop codon of CP and resulted in the expression of the target gene as a readthrough CP-fused protein. However, the T7-promoter-driven infectious clone used in these studies needs in vitro transcription to obtain the viral RNA transcripts then mechanically inoculated into muskmelon, which makes it inconvenient to use [36, 37]. In 2015, Zheng et al. developed a CGMMV infectious clone tagged with GFP under the transcriptional control of the CaMV 35S promoter, which can establish infection in cucumber (Cucumis sativus), watermelon (Citrullus lanatus), bottle gourd (Lagenaria siceraria), and Nicotiana benthamiana plants via agroinoculation [41]. The GFP coding region was placed in frame between the coding sequences between MP and CP. They chose 234 nt before the CP initiation codon as the CP 5′ subgenomic promoter based on their rapid amplification of cDNA ends (RACE) result and the sequences of CP SGP of TMV and constructed three vectors with different lengths of the 5′-proximal CP ORF to drive GFP expression, which successfully infected bottle gourd and displays strong green fluorescence in symptom leaves. However, GFP was easily deleted from the viral genome due to the recombination of duplicated SGP along with infection [41].

Shortly after that, Liu et al. developed a CGMMV-based VIGS vector that silences PDS in watermelon, melon, cucumber, and bottle gourd. They adopted a similar strategy with TMV VIGS vectors previously reported to generate a series of CGMMV VIGS vectors, which explored different lengths of CP SGP. Their results indicated that the PDS gene fragments induced a more robust silencing phenotype when the vector contained a 190-bp duplicated copy of the putative CP SGP. The length and structure of the insert sequence could affect the silencing efficiency. Their results showed that the CGMMV vectors harboring the PDS gene sequence of 300 bp in length had the highest silencing efficiency in cucurbits, and the silencing phenotypes could persist for at least one month [42]. Up to now, the CGMMV-based VIGS system has been widely used in cucurbits studies, such as the tolerance to salinity stress, chilling tolerance, and functional analyses of resistance-related genes [43, 44, 45].

3.2 Cucumber fruit mottle mosaic virus

Cucumber fruit mottle mosaic virus (CFMMV), which is also a member of the genus Tobamovirus, mainly infects cucurbits. As CGMMV, CFMMV has also been constructed as virus vectors for functional genomic studies in cucurbits. In 2016, Rhee et al. constructed a CFMMV vector, which could express enhanced green fluorescent protein (EGFP) in cucumber, melon, and watermelon. They tested the active region of CP SGP and found that the region from −55 to +100 nt was identified as the active core promoter. Then, they chose CP SGP with 93 nt before the CP initiation to express the CP of CFMMV, and CP SGP with 100 nt to drive the EGFP expression. The EGFP can be expressed in hypocotyl and leaf veins, but the expression level is low in mesophyll cells. They also found that co-infiltration with the P19 RNA silence suppressor could enhance EGFP expression [38].

Recently, Rhee et al. further constructed an efficient CFMMV VIGS vector that exhibits high gene silencing efficiency and long-lasting PDS-silenced phenotype in cucurbit plants. The CFMMV VIGS vector adopts a similar strategy as the CFMMV overexpression vectors they previously developed. They found that the best vector to express EGFP was also the best VIGS vector to silence PDS. The vector could effectively silence the corresponding PDS gene in melon, cucumber, and watermelon, resulting in photobleaching in leaves and reproductive organs. It is worth noting that they used this optimized system to identify genes involved in male sterility, which suggests that VIGS in watermelon is functional [46].

Although some tobamoviruses vectors show significant overexpression and silencing effects in cucurbits soon after inoculation, the repeated CP SGP sequences would result in homologous recombination. In the future, the effect of CGMMV-based gene overexpression and silencing will be improved by replacing the native SGP of CGMMV CP with the SGP of other tobamoviruses CP to stabilize the inserted fragments.

4. Apple latent spherical virus

Apple latent spherical virus (ALSV) is a member of the genus Cheravirus (family Comoviridae), which is known for the presence of atypical members. ALSV contains two single-stranded RNA (ssRNA) species and three capsid proteins. RNA 1 has a single ORF encoding a polypeptide, which contains the consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine, protease, and the RNA polymerase. RNA 2 also has a single ORF encoding a polypeptide of 119 K/108 K containing a 53 K/42 K movement protein on the N-terminal side and three capsid proteins (Vp25, Vp20, and Vp24) [47].

In the early period, researchers constructed ALSV vectors to express GFP allowing us to trace the cell-to-cell and long-distance movement of ALSV in infected plant tissues of N. benthamiana [48, 49]. As ALSV is a kind of latent virus and does not induce any obvious symptoms in most host plants, it can be used for functional genomics in the host plants. This is one of the requirements for plant virus vectors used for VIGS [50]. ALSV was developed into VIGS vectors by Igarashi et al. in 2009. The target gene fragment can be inserted between 42KP and Vp25. The ALSV vector induced highly uniform phenotypes induced by knocking down the subunit of magnesium chelatase (SU) and PDS in plants, including cucurbit species such as cucumber, watermelon, zucchini (Cucurbita pepo), loofah, and bottle gourd. ALSV-based VIGS vector can induce a highly obvious and stable gene-silenced phenotype throughout plant growth in infected plants. Recently, they further developed an efficient virus-induced gene silencing system using ALSV in pumpkins (Cucurbita spp.) [51].

However, ALSV vectors have several limitations, such as the ALSV vector is difficult to use for high-throughput functional genomics since ALSV proteins are expressed by polyprotein proteolytic processing and the inserted gene sequences cannot produce stop codon in the open reading frame of ALSV. Another disadvantage is that ALSV-cDNA clones are less efficient for direct inoculation into host plants [52].

5. Potyviruses

Potyvirus is a genus that belongs to the family Potyviridae. Potyviruses exist as flexuous rod-shaped particles ranging from 720 to 900 nm in length with an ssRNA genome of approximately 10 kb. Potyviruses have only an ORF encoding a large polyprotein, which is processed into ten functional proteins by three viral proteinases [53]. P3N-PIPO protein is produced using a viral polymerase slippage mechanism [54]. Now, lots of potyviruses have been developed into infectious clones for potyviral studies expression vectors [54]. The foreign gene fragments are usually inserted between the first protein (P1) and helper component-proteinase (HC-Pro) or between nuclear inclusion protein b (NIb) and CP cistrons [55, 56]. Potyviruses are considered promising expression vectors since their proteolytic processing strategy of gene expression ensures that the foreign protein can be produced in equal amounts with viral proteins [57]. Compared with other known viral vectors that cause severe symptoms to host plants, attenuated mutants with a mutation in HC-Pro, which is both a major virulence determinant of potyviruses and RNA silence suppressor, make it possible for the potyviruses to be the environmentally safe virus vector for the expression of various foreign genes in plants [58].

5.1 Zucchini yellow mosaic virus

Zucchini yellow mosaic virus (ZYMV), which belongs to the genus Potyvirus, has been used as expression vector in cucurbit plants for a long time. In 2001, Arazi et al. exploited ZYMV to develop a novel virus-based vector system for the expression of foreign genes in cucurbits. The virus vectors in this study were generated from an attenuated mutant (AG) [57], which accumulates to the same virus accumulation level as the severe ZYMV strain in cucurbits, without eliciting any phenotypic and developmental impairment [59]. This vector could express GFP, uidA (β-glucuronidase; GUS), and human interferon-alpha 2 (IFN) in leaves, stems roots, male flowers, and fruits of squash (Cucurbita spp.) or cucumber. In the same year, they further expressed the bar gene using this vector in melon, cucumber, squash, and watermelon, which confer these plants’ resistance against glufosinate ammonium-based herbicides [60]. In 2006, this vector was used both for overexpression- and down-regulation of Trichoderma-induced MAPK (TIPK) gene expression in cucumber [61]. In 2016, Kang and coauthors expressed GFP and bar in zucchini by simple rub-inoculation of plasmid DNAs of the ZYMV-based expression constructs [62].

5.2 Watermelon mosaic virus

Watermelon mosaic virus (WMV) is also a member of the genus Potyvirus, which exhibits a wide host range and is one of the most prevalent viruses in cucurbits worldwide [63]. In 2019, Aragonés and coauthors characterized a WMV isolate that induces mild symptoms in cucurbits [64], and then they developed a VIGS vector based on the mild isolate. Melon PDS fragments were inserted between NIb and CP in the form of sense, antisense, and hairpin. PDS fragments in sense and antisense orientations were more stable in the viral progeny, and the sense construct triggered the best silencing effect compared to those constructs with antisense and hairpin fragments. They further confirmed this result by lytargeting melon Magnesium chelatase subunit I (CHLI) using the VIGS vector. Silencing of CHLI can be easily tracked by a foliar decoloration phenotype, as a consequence of deficient chlorophyll accumulation, which exhibited decolored spots. The results showed that the VIGS vector constructed based on the mild isolate WMV could be a useful tool in cucurbit gene studies [65]. As with other potyviruses, WMV could also be used as an expression vector in cucurbits [58, 66].

Potyviruses are the largest group of plant RNA viruses, with approximately 200 species [67]. However, relatively few VIGS vectors have been derived from potyviruses, as potyviruses possess a strong RNA silencing suppressor that usually precludes VIGS. Some nonsynonymous mutants in HC-Pro can abolish HC-Pro’s RNA silencing suppression (RSS) activity [67]. Therefore, a reliable potyvirus-based VIGS vector could be developed by introducing site-directed mutations to the HC-Pro gene, which attenuates virus virulence [65].

6. Tobacco rattle virus

Tobacco rattle virus (TRV) is one of the members of the genus Tobravirus. The genome of TRV is divided into two positive-sense, ssRNAs, each of which is encapsidated separately into rod-shaped particles. The genome of TRV RNA1 encodes replicase, 1a and 1b, which is sufficient for replication and movement within the host plants. The genome of RNA2 encodes CP, which can produce virus particles, and allows nematode-mediated transmission between hosts [68, 69]. TRV induces very mild symptoms and is suitable for gene silencing in most plants. The nonessential 29.4 k and 32.8 k genes of RNA2 can be replaced with an MCS for the insertion of the target gene sequence [68].

In 2019, a TRV-VIGS system was applied in cucumber using a new infection method with a special agroinfiltration solution, which provides an efficient and easy method for functional analysis of genes in cucumber [70]. PDS and glycerol-3-phosphate 2-O-acyltransferase 6 (GPAT6) gene were successfully silenced with this VIGS system in cucumber. The silencing of GATP6 resulted in the resistance to autotoxin stress mimicked by cinnamic acid (CA) in cucumber. This TRV-VIGS system enables efficient silencing of cucumber genes at the whole-plant level without time-consuming transformation or manipulation [71]. In the same year, Liao et al. developed transient overexpression and VIGS systems for oriental melon using the sprout absorption method. They successfully overexpressed and silenced candidate lipoxygenases (LOXs) gene in oriental melon using TRV-based VIGS they modified [72]. The overexpression of CmLOX10 could trigger cell death responses in oriental melon. The disadvantage of the TRV-based VIGS vector is that, it’s difficult to infect cucurbits via traditional inoculation method. The infection method used in these researches was established based on the whole cotyledonary node method of regeneration described by Ma and Wu [70, 73], which is operation complexity and time-consuming.

7. Cucurbit chlorotic yellows virus

Cucurbit chlorotic yellows virus (CCYV) is a recently discovered cucurbit-infecting virus, which belongs to the Crinivirus genus in the family Closteroviridae. CCYV has characteristic long, flexuous rod-shaped virions, ranging in size from 700 to 900 nm or 650–850 nm. The genome of CCYV contains two RNAs. RNA1 includes two ORFs, ORFs 1a and 1b, which encode papain-like cysteine proteinase (PRO), methyltransferase (MTR), helicase (HEL), and RdRp, respectively. RNA2 includes seven ORF that individually encodes CP (major coat protein), CPm (minor coat protein), 70-kDa heat shock proteins (Hsp70h), and P59, which are essential for virion structural components, movement, and vector transmission. CCYV causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves [74]. Respectively, Wei et al. constructed a CCYV vector for GFP expression. They inserted different subgenomic RNA promoters and a GFP coding sequence into the upstream of the CP coding sequence to initiate the expression of GFP and CP expression. The GFP fluorescence was only detectable in cucumber leaf veins and surrounding cells [74]. As the infection of CCYV is limited in the phloem, CCYV-based vectors are specifically useful for studying the function of gene localized in the phloem [75].

8. Bean yellow dwarf virus

Bean yellow dwarf virus (BeYDV) belongs to the Mastrevirus genus of the family Geminiviridae. BeYDV comprises monopartite or bipartite circular single-stranded circular DNA (ssDNA) viruses characterized by their germinate particles comprised of two joined incomplete icosahedra [76]. The circular ssDNA of BeYDV is 2.5–2.7 kb in length and encodes MP, CP, and replication associated proteins A and B (RepA and RepB) [77]. BeYDV uses a rolling circle mechanism to replicate its genome, resulting in a very high yield of copies, which has been used to boost protein expression in transgenic plants and for efficient transient expression of foreign protein plants [78, 79]. BeYDV has a broad host range in plants, including many cucurbits. Yamamoto et al. developed a transient expression system that combines geminiviral replication and a double terminator, which could improve the protein expression in melon. In particular, the expression level was the highest when the HSP and Ext terminators were used as double terminators [80]. However, this vector cannot infect melon systemically, which could only be used for transient expression.

9. Conclusion

Most cucurbit plants possess very low transformation efficiency [81]. Plant virus-based tools have been used in gene silencing and overexpression in cucurbits. Up to now, TRSV and ALSV vectors, which have been used in different research groups, display good performance in cucurbit plants [16, 28, 29, 51, 82]. The efficiency of other virus vectors infecting cucurbit plants is relatively low. However, the VIGS capacity of CFMMV-based vectors has been enhanced recently, which provides guidance for the modification of other viral vectors [46]. In addition, VIGE technologies have been developed to overcome the bottleneck caused by tissue culture [24]. Some plant viruses mentioned in this review that infect cucurbits such as BeYDV and TRV have been harnessed for VIGE in N. benthamiana, Arabidopsis thaliana, potato (Solanum tuberosum), and tomato (Solanum lycopersicum) [83, 84, 85, 86]. However, these VIGE tools have not been implemented in cucurbit crops yet. TMV has been constructed to deliver biologically functional single guide RNAs (sgRNAs) through the CP subgenomic promoter in N. benthamiana in 2017 [22]. As members of Tobamovirus, CGMMV and CFMMV also have the potential of delivering sgRNA. Because the correlation between the stability of virus vectors and the size of foreign genes is negative, it is difficult for most plant viruses to directly deliver large proteins such as Cas9 [87]. Up to now, most types of plant virus vectors were used to deliver gRNAs into Cas9-overexpressed plants for VIGE [24]. However, TRSV-based vectors, which have displayed powerful capacity to knock down genes and miRNAs in cucurbits, were recently reported to express SaCas9 in N. benthamiana without comprising virus stability [16]. Therefore, with the development of biology technology, plant virus-based tools will further facilitate the study of cucurbit plants.

Acknowledgments

This study was funded by National Natural Science Foundation of China (NSFC, 31801704), China Postdoctoral Science Foundation (2021 M691973; 2022 T150389), and Research and Development Program in Shandong Province (2021LZGC015).

Conflict of interest

The authors declare no conflict of interest.