Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots

Abstract Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof‐of‐concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus‐induced gene silencing (VIGS) and virus‐mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus‐induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize–microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize–microbe interactions and more broadly in the context of the monocot functional genomics toolbox.

for modifying genes associated with host-pathogen interactions in the future (Gentzel et al., 2022).
Zea mays (maize) is a key cereal crop that is grown worldwide and is an important model species. There are many genetic and genomic resources available to facilitate gene function analyses, and over the years a growing number of virus-based vectors have been reported to be useful in maize. At the present time, there are at least 11 different virus species that have been tested in maize for their utility in VIGS, VOX, and/or VIGE (Table 1). We also consider another use, virus-induced flowering (VIF), which has not yet been demonstrated in maize, but is feasible in other monocots. VIF results from the transient overexpression of Flowering Locus T (FT) homologues in plants by means of a virus, and it is proposed to be of potential use to accelerate breeding programmes through the induction of flowering (Ayre et al., 2020;Yuan et al., 2020).
Of the 11 virus species for which there is evidence that they may have utility as viral vectors in maize, each one has inherent advantages and disadvantages. Some viruses, such as cucumber mosaic virus (CMV), brome mosaic virus (BMV), and maize rayado fino virus (MRFV) (Ding et al., 2006;Mlotshwa et al., 2020;Wang et al., 2016), only have capacity to carry relatively small foreign inserts and therefore their use will probably be limited to VIGS and potentially VIGE (Willemsen & Zwart, 2019). Tobacco rattle virus (TRV), which is exceptionally useful in many dicots (Shi et al., 2021), was reported to cause VIGS of phytoene desaturase (ZmPds) in maize seedlings (Zhang et al., 2017), but the question of how well it can actually replicate and move systemically in maize has not been adequately addressed. Sugarcane mosaic virus (SCMV), maize dwarf mosaic virus (MDMV), and wheat streak mosaic virus (WSMV) are potyviruses that encode a large polyprotein. Protein expression in maize has been demonstrated via insertion of cloning sites that allow these viruses to express proteins from sequences that are cloned in frame with the viral polyprotein (Mei et al., 2019;Tatineni et al., 2010;Xie et al., 2021). Interestingly, gene fragments for VIGS applications can also be inserted into these positions as long as the open reading frame (ORF) is preserved (Chung et al., 2022;Xie et al., 2021).
Moreover, it was shown that MDMV can be used to simultaneously express a protein and silence multiple target genes in maize plants . Because SCMV, MDMV, and WSMV encode large polyproteins, their genomes also serve as the sole viral messenger RNAs and therefore they do not produce shorter subgenomic messenger RNAs. The lack of subgenomic messenger RNA suggests that these viruses may not be useful for delivering guide RNAs for VIGE, but there may be strategies to overcome this limitation .
The viruses mentioned so far have single-stranded, positivesense RNA genomes, and for these kinds of viruses the technology to produce infectious clones and manipulate them to accept the insertion of foreign sequences has been available since the 1980s (e.g., Ahlquist et al., 1984;French et al., 1986). Maize mosaic virus (MMV) and barley yellow striate mosaic virus (BYSMV) in contrast are negative (−)-strand RNA viruses, and only recently has the ability to engineer infectious clones derived from them been demonstrated (Gao et al., 2019;Kanakala et al., 2022). These (−)-strand RNA viruses are interesting because they have more stable insertions that are less susceptible to homologous recombination and spontaneous deletions, and they independently express multiple sequences, including ORFs, gene fragments, and guide RNA. As such, there is anticipation over their use for delivering clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) reagents as well as VIGS and VOX of multigenic metabolic pathways.
If technical hurdles can be overcome related to initiating infections, which currently rely on inoculation into transgenic plant lines expressing the replication proteins and subsequent transfer of the resulting virions into experimental host plants by way of an insect vector, then these viruses may gain widespread use.
At the present time, the virus species that is most widely used in maize for gene function analyses is foxtail mosaic virus (FoMV).
FoMV is becoming routinely used for VIGS and VOX, and it can deliver functional guide RNAs that can direct genome edits in maize TA B L E 1 Viral vectors developed for use in Zea mays.

| FoMV IS A P OTE X VIRUS WITH A WIDE HOS T R ANG E
FoMV was first identified by the mild chlorotic mosaic symptoms it caused on the leaves of Setaria viridis (green foxtail) in a field in Kansas, United States (Paulsen & Niblett, 1977). FoMV is particularly interesting because it has a large experimental host range, infecting 56 monocot species and 35 dicot species, including numerous graminaceous species such as maize, Hordeum vulgare (barley), Sorghum bicolor (sorghum), Setaria spp.
(millets), and Triticum aestivum (wheat) (Paulsen & Niblett, 1977). Despite its wide host range and ability to naturally infect weedy and crop hosts in the field (Paulsen & Niblett, 1977;Seifers et al., 1999), it has not been associated with major disease outbreaks or yield losses. Foxtail mosaic virus belongs to the genus Potexvirus, of which several species have been developed as viral vectors (Abrahamien et al., 2020). Potexviruses have been used as viral vectors due to their small but modifiable genomes, ability to spread systemically, and broad host range.
The genome of FoMV was first sequenced and published in 1991 and revised in 2008 after infectious full-length clones were generated and sequenced (Bancroft et al., 1991;Bruun-Rasmussen et al., 2008). Like other potexviruses, FoMV has a single-stranded, positive-sense RNA genome that is 6.2 kilobases (kb) in length. It as well as providing movement functions. Lastly, ORF 5 produces the coat protein (CP) from sgPro2, which is necessary for virion assembly and long-distance movement (Candresse et al., 2012). Unlike other potexviruses, an ORF 5A was also identified in the FoMV genome ( Figure 1a), but mutations disrupting the start codon showed that it is dispensable for infection and its disruption had no impact on viral replication and fitness (Mei et al., 2019;Robertson et al., 2000).

| ENG INEERING FoMV FOR VI G S , VOX , AND VI G E
Potexviruses have been good candidates for development into viral expression vectors due to their ability to accept a duplicated subgenomic promoter and lack of theoretical virion size constraints due to being rod-shaped, filamentous viruses (Willemsen & Zwart, 2019). Potato virus X (PVX) is the archetypal potexvirus that was first engineered for transient gene expression in plants (Chapman et al., 1992). Initial PVX expression vector designs replaced the CP with the coding sequence of the marker protein, β-glucuronidase (GUS). While GUS was highly expressed, this strategy prevented systemic movement of the virus (Chapman et al., 1992). A second strategy duplicated the CP promoter (sgPro2) and inserted it between the TGB3 and CP ORFs. The GUS coding sequence was fused to the duplicated sgPro2 promoter, and this recombinant PVX successfully expressed GUS systemically and set a precedent for designing potexvirus expression vectors (Chapman et al., 1992).
Subsequently, the sgPro2 was duplicated in a way that preserved the ORF 5A, and cleavage sites for the HpaI, MluI, XhoI, and AscI restriction enzymes were inserted just after the duplicated sgPro2. To generate inoculum, this clone is agroinfiltrated into N. benthamiana, and then the infected leaves are used to inoculate monocot plant species:

F I G U R E 1 Schematic representations of foxtail mosaic virus (FoMV) and vectors that have been derived from it. (a)
Wild-type FoMV from left to right, the oval represents the 5′ 7-methylguanosine cap structure, followed by a 5′ untranslated region (UTR), the RNA dependent RNA polymerase (RDRP), subgenomic promoter 1 (sgPro1) driving transcription of subgenomic RNA 1, the triple gene block proteins (TGB) 1, 2, and 3, a predicted open reading frame (ORF) 5A (5A) that is unnecessary for infection (Robertson et al., 2000), subgenomic promoter 2 (sgPro2) driving the transcription of subgenomic RNA 2, the coat protein (CP), a 3′ UTR that terminates at a polyA tail.  2016), and it includes a DP to drive expression of a GOI or produce functional single-guide RNAs (gR) for CRISPR/Cas9 gene editing applications. All of the FoMV viral vector designs (b-f) are transcribed under the control of a 2× cauliflower mosaic virus 35S promoter (35S) and the nopaline synthase (NOS) terminator, and the grey arrows located along the genomes represent the positions of the sgPro1, sgPro2, and DPs.
In particular, Agrobacterium-based methods that deliver engi- with FoMV or SCMV. This method was inspired by classic work with infectious clones of maize streak virus, a geminivirus, that could be inoculated into maize by injecting the Agrobacterium strains into the whorl of seedlings 2-3 mm above the shoot apical meristem (Grimsley et al., 1986). This method can be applied for both FoMV VIGS and VOX applications, but there may be a negative correlation between insert size and inoculation efficiency (Beernink et al., 2021).

| E XPERIMENTAL DE S IG N CONS IDER ATIONS
In addition to inoculation methods, we provide some key considerations to aid in the design and interpretation of experiments using ing 329-and 313-nt gene fragments were similar to the empty vector but much higher than FoMV-GFP, which carries a 711-nt insertion.
The effectiveness of VIGS and VOX can also be altered by the stability of the insert. For example, a 300-nt insert targeting sorghum phytoene desaturase (SbPds) was stably maintained at 21 days postinoculation (dpi) in 72%-90% of plants, but a 300-nt insert targeting ubiquitin (SbUb) was stably maintained in only 36%-45% of the plants in sorghum genotype BTx623 . Interestingly, in the sorghum genotype BTx430, the SbPds gene fragment was stably maintained in FoMV in 100% of the plants screened, and the SbUB fragment was stable in only 12%-25% of the plants. These data show that the insert sequence and host genotype can influence insert stability. However, time after inoculation is also critical. The retention of the SbPds fragment was similar at 14, 21, and 28 dpi, and the SbUb insert was stable at 14 dpi but became increasingly unstable at 21 and 28 dpi.
These data from sorghum are consistent with prior results from maize using a ZmPds insert (Mei et al., 2016). In plants that were inoculated at 7 days after sowing, the ZmPds insert was stably retained in leaves 4-6 but as later leaves developed, the insert in FoMV became increasingly unstable. By leaf 9, ZmPds was beginning to be deleted and was

| APPLI C ATI ON S OF FoMV VEC TOR S IN UNDER S TANDING OF MAIZE-MICROB E INTER AC TI ON S
To date, the primary application of FoMV vectors has occurred in topics related to maize-microbe interactions (Table 2). Here, we highlight their application in some of the studies focused on maizemicrobe interactions that have benefited from the availability of these resources for investigating the functions of both host and pathogen genes. Plants can recognize the presence of pathogens through the action of pattern-recognition receptors that activate pattern-triggered immunity (PTI) in response to conserved molecular features, such as flagellin (flg22 peptide, bacteria) or chitin (fungi) . Activation of PTI is accompanied by a variety of changes, including reactive oxygen species (ROS) burst, callose deposition, and increased expression of defence genes. Successful pathogens secrete effectors that inhibit PTI by targeting different proteins involved in regulating or mediating it (Toruño et al., 2016).
Resistance proteins recognize the presence of effectors, either directly or indirectly, and activate effector-triggered immune responses (ETI) that often result in hypersensitive cell death (HR) (Cui et al., 2015). Much remains to be learned about the regulation of PTI, ETI, resistance protein function, and pathogen effector functions in maize, and VIGS and VOX approaches are contributing key information that is helping to advance understanding of these various facets of maize-microbe interactions. FoMV has also been used to ectopically express U. maydis effectors as fusion proteins with epitope tags. In three different expression studies, the Bouton et al. (2018) PV101 vector was used to express effectors lacking their signal peptides that were fused to the myc or HA epitopes (Darino et al., 2021;Navarette et al., 2021;Saado et al., 2022) ( Table 2). The effector fusions were co-expressed with the p19 protein, which is a suppressor of RNA silencing that promotes accumulation of the recombinant viruses. The mCherry protein was also co-expressed with p19 and the effector fusion in two of the studies, which provides a non-destructive reporter on virus accumulation and spread. Ectopic expression of the effectors enabled analysis of their roles in promoting cell death or suppressing host defences.

| Use of FoMV vectors to investigate genes involved in maize immunity
FoMV VIGS has been used to investigate the functions of maize genes from the perspectives of resistance protein signalling and TA B L E 2 Foxtail mosaic virus used as a viral vector in plants.  Table 2). One construct targeted ZmFLR1 and ZmFLR2, and a second one targeted ZmFLR3. ZmFLR1 and ZmFLR2 had to be co-silenced due to high sequence homology.
To separately silence ZmFLR1/2 and ZmFLR3, it was necessary to target the least conserved region encoding the transmembrane domain. VIGS of ZmFLR1, 2 and 3 reduced their expression by approximately 65%-70%, and ROS production was dramatically reduced in response to flg22 and chitin application to leaf discs. Plants in which ZmFLR1/2 or ZmFLR3 were silenced were challenged with four different fungal pathogens, and disease severity increased for all providing evidence that the ZmFLRs have important roles in maize antifungal immune responses.
In the largest-scale VIGS study in maize to date, Murphree

Maize chlorotic mottle virus (MCMV) causes maize lethal necrosis
when it co-infects maize along with unrelated viruses, such as potyviruses like SCMV (Redinbaugh & Stewart, 2018). Jiao et al. (2021) were interested in the molecular mechanisms underlying the pathogenicity of MCMV, and found some preliminary evidence that its 31 kDa protein (p31) was a major pathogenicity determinant. They expressed individual MCMV proteins p31, p7a, and the readthrough domain (RTD) or a GFP control fused to a 3× FLAG tag from the PV101 FoMV vector in B73 maize seedlings. FoMV expressing p31 or the RTD induced necrotic lesions on maize leaves but p7a and the GFP control did not. These data demonstrated that the RTD portion of p31 is responsible for the necrosis induced by MCMV infection.
Subsequently, they showed the FoMV expressing p31 suppresses salicylic acid (SA) production as well as the expression of PR genes when co-inoculated with MCMV. These results showed that p31 suppresses the SA-mediated defence responses induced by MCMV.

Xu et al. (2022) used the Liu et al. (2016) FoMV vector to silence
ZmTGL, which codes for the production of triacylglycerol (Table 2).
ZmTGL was identified as interacting with SCMV's helper component proteinase (HC-Pro) through protein pull-down and tandem mass spectrometry. Silencing ZmTGL1 reduced its mRNA transcripts by 50% and resulted in a 2-3-fold greater accumulation of SCMV. This silencing phenotype is consistent with a role for ZmTGL1 in reducing the accumulation of SCMV HC-Pro, which is a silencing suppressor required for efficient replication of SCMV.

| FoMV AND VI G E
Engineering viruses to deliver gene editing components has been a rapidly expanding area of research. The use of viral vectors overcomes bottlenecks associated with traditional transgenesis methods that are needed to introduce gene editing reagents into plants (Yin et al., 2017). Virus-based delivery of gene editing reagents can potentially open access to gene editing or enhance gene editing efficiency in many plant species without the need to go through the processes of transformation and regeneration (Scholthof et al., 1996).
Targeted gene editing technologies have revolutionized genetics over the past decade. Meganucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) are all genome editing platforms with a high level of target specificity, but they are limited by challenges in modifying that specificity (Voytas & Gao, 2014). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated proteins (e.g., Cas9) have been harnessed to activate, suppress, delete, and add new target genes in the genomes of many organisms (Belhaj et al., 2015;Bortesi & Fischer, 2015;Doudna & Charpentier, 2014;Pennisi, 2013). CRISPR-based genome editing technologies continue to be developed and improved for new applications as well as increased efficiency and target specificity.
The utility of these systems is the ease with which they can be reprogrammed through the delivery of specific single-guide RNAs (sgRNAs). Several plant viruses with positive-sense RNA genomes that had previously been used for VIGS and/or VOX were demonstrated to also deliver sgRNAs systemically and induce edits in host plants that express Cas proteins. Some of the first viral systems established to deliver sgRNAs and successfully validate gene editing include tobacco rattle virus (TRV) (Ali, Abul-faraj, Li, et al., 2015;, tobacco mosaic virus (TMV) (Cody et al., 2017), and pea early browning virus (PEBV) (Ali et al., 2018). Gene editing as a result of virus-delivered sgR-NAs predominantly occurs in somatic cells, but some viruses expressing sgRNA have been shown to efficiently induce heritable genome edits, such as TRV and PVX in N. benthamiana and BSMV in wheat Ellison et al., 2020;Li et al., 2021;Uranga et al., 2021). In the case of TRV, the efficiency of inducing heritable genome edits is augmented significantly if a mobile RNA sequence, such as Arabidopsis FT, is fused to the sgRNA Ellison et al., 2020). However, for BSMV, the addition of RNA mobility sequences hinders the ability of the virus to induce heritable genome edits in wheat (Li et al., 2021).
FoMV clones carrying sgRNA were able to induce somatic genome edits in the Pds gene of N. benthamiana plants expressing Cas9. The induced mutations were small insertions and deletions (indels), and they occurred in leaves and flowers over the course of plant development (Mei et al., 2019). However, the level of mutation was not sufficient to cause the photobleaching phenotype expected for Pds loss of function and heritable mutations were not observed, which is in contrast to TRV, PVX, and BSMV in N. benthamiana .
Interestingly, the frequency of mutations induced by FoMV express- In maize, the frequency of genome editing in leaves was relatively low compared to N. benthamiana and S. viridis, and heritable mutations were also not observed  Moreover, the studies presented show that FoMV VIGS and VOX can be used successfully to investigate resistance gene function, PTI, and maize-fungus and maize-virus interactions. We also anticipate that it will be useful to investigate maize-bacteria interactions, although this is yet to be demonstrated. For example  showed that FoMV VIGS of receptor-like cytoplasmic kinases in sorghum suppressed basal immune responses rendering the plants more susceptible to bacterial pathogens. Similarly, FoMV VOX of fungal effector proteins and viral proteins has been very useful for exploring maize-microbe interactions, and we would anticipate that this would be the case for effectors encoded by bacteria, nematodes, and insects as well. So far, VIGS and VOX have been demonstrated to work in leaves and switchgrass roots (Tiedge et al., 2022), and so it will be interesting to see if these approaches can be applied to other organs in the future. In addition, it will be interesting to determine if FoMV can be used as a vector for host-induced gene silencing (HIGS) to knockdown the expression of genes of pathogens as they attempt to infect plants in which FoMV carrying fragments of pathogen genes are replicating, as has been shown for other viruses (Hu et al., 2020;McCaghey et al., 2021;Nowara et al., 2010;Panwar et al., 2013;Yin et al., 2015).
It is exciting to see that FoMV-based resources and their corresponding protocols are being adopted successfully by many laboratories. While FoMV has been applied mainly for research in maize-microbe interactions at this time, we anticipate that it will be useful in studying the functions of genes involved in other aspects of maize biology. A major rationale for engineering FoMV for VIGS and VOX applications was its reportedly broad host range, particularly in monocots (Scofield & Nelson, 2009), and several recent publications suggest that FoMV is meeting expectations ( Library.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created.