A fast and efficient method to introduce apple latent spherical virus to legume plants via Agrobacterium rhizogenes‐mediated transformation of hairy roots

Virus‐induced gene silencing (VIGS) is a functional genomics tool used to determine the function of unknown genes or assess the impact of gene silencing on plant phenotype. However, VIGS methods for analyzing gene function are not equally efficient across species, and virus inoculation is difficult for some legume species. We describe a fast and efficient inoculation method using Agrobacterium rhizogenes K599 harboring an apple latent spherical virus (ALSV) full‐length cDNA clone. Pisum sativum cv. AAC Lacombe and Lens culinaris cv. CDC Viceroy showed silencing rates of 100% and 47.7%, respectively, within 30 days starting from seed. To our knowledge, this is the first report of virus inoculation by A. rhizogenes‐mediated introduction to legume plants. This work paves the way for high throughput gene function screening in Pisum sativum and other closely related legume crops using ALSV as a symptomless VIGS vector.


| INTRODUCTION
Legume crops, including pea and lentil, have a beneficial nutrition profile for human and animal consumption. Many studies of legume plants have focused on enhancing sustainable production and improving agronomic traits of interest (Varshney et al., 2009), and genes potentially associated with essential agronomic traits have been recognized and will continue to be identified with the sequencing of legume plant genomes (Rispail & Rubiales, 2016;Schmutz et al., 2010). In addition to the use of these resources in genomicsassisted breeding, functional genomics tools allow for characterization of gene function and identification of molecular interactions beyond inference based on sequence alone.
Virus-induced gene silencing (VIGS) has been used to characterize gene function. Tobacco rattle virus (TRV) has been employed as a VIGS vector in Nicotiana benthamiana, Arabidopsis thaliana, and tomato successfully (Burch-Smith et al., 2006;Liu et al., 2002Liu et al., , 2004. Clover yellow vein virus (pCIYVV), pea early browning virus (PEBV), bean pod mottle virus (BPMV), and cucumber mosaic virus (CMV) have all been developed as silencing vectors for legume species (Constantin et al., 2004;Gronlund et al., 2010;Luo et al., 2013Luo et al., , 2016Masuta et al., 2000;Nagamatsu et al., 2007;Zhang & Ghabrial, 2006). However, limited host ranges and phenotypic symptoms associated with the virus itself has restricted the utility of VIGS as a functional genomics tool in this group of plants. Apple latent spherical virus (ALSV) has been used as a gene silencing vector in a broad range of plants including tobacco, tomato, Arabidopsis, cucurbits, and legumes.
It is a bipartite RNA virus that relies on transcription of a multi-protein polypeptide that is then processed into catalytic and structural virus proteins (Igarashi et al., 2009;Li et al., 2000;Sasaki et al., 2011). Igarashi et al. (2009) used biolistic (particle bombardment) inoculation to introduce RNA from ALSV infected quinoa to silence phytoene desaturase (PDS) in soybean, pea, adzuki bean, and cowpea (Igarashi et al., 2009). Agrobacterium-mediated transformation is often inefficient for legume plants. Common methods to introduce ALSV to soybean, common bean, and pea were less effective than in other species, although ALSV has been successfully introduced in pea plants by stem injection of Agrobacterium tumefaciens (Xiong et al., 2019).
This led us to consider induction of hairy root culture and introduction of ALSV using Agrobacterium rhizogenes.
Agrobacterium rhizogenes-mediated transformation of legumes was demonstrated in Lotus corniculatus, Phaseolus vulgaris, Medicago sativa, and Glycine max (Kereszt et al., 2007). A. rhizogenes K599 is a strain able to induce hairy roots on soybean and common bean explants (Kereszt et al., 2007;Khandual & Reddy, 2014). To extend the application of ALSV as a gene silencing vector in legumes, we tested the infection and silencing efficiency of K599 to introduce ALSV infectious clones to legume plants (soybean, common bean, lentil, and pea). To our knowledge, this is the first report of infectious clone inoculation and VIGS by hairy root transformation using A. rhizogenes.

| Pisum sativum PDS cloning and constructs
PsPDS fragment (336 bp) was amplified from P. sativum cv. Lacombe cDNA using a primer pair (forward primer: 5 0 -CACACACTCGAGA GCAGAAGCCCCCTTCTGAG-3 0 with XhoI underlined and reverse primer: 5 0 -CACACAGGATCCTTGTTTTGTGTAATCTCCTG-3 0 with BamHI underlined). The PsPDS PCR product was cloned into pCR Blunt II TOPO (Zero Blunt PCR Cloning Kit, Thermo Fisher Scientific) for sequencing. Following sequence confirmation, the fragment was released by digestion with XhoI and BamHI and inserted into the same sites of pBICAL2 (Kaido et al., 2014) to produce pBICAL2-PsPDS. and germinated in the dark at 25 C. Three days after incubation, seeds were rinsed with sterile water, and the seedcoats were removed. Seedlings with a radicle between 2 and 3 cm were selected, and the hypocotyl was cut below the cotyledonary node with a scalpel. The radicle was removed, and exposed hypocotyl was immersed in the A. rhizogenes cell suspension described above for 2 min. The

| Agrobacterium rhizogenes K599 transformation of ALSV and PDS silencing in L. culinaris
We used the PsPDS construct to test this protocol in lentils. Using the same transformation protocol, germinated lentil seeds were severed and coated with K599 strains carrying pBICAL1 and pBICAL2-PsPDS.
Again, a mix of K599 carrying pBICAL1 and pBICAL2 served as a control. Forty-nine percent of the lentil seeds incubated produced hairy roots. All seedlings with hairy roots were transplanted to soil and grown. Thirty days post transformation, 47.7% (3/8, 5/9, 5/10) exhibited PDS silencing (Figure 2c). ALSV RNA1 fragments were present in plants transformed with empty vectors as well as in PDS silenced plants (Figure 2d).

| DISCUSSION
Agrobacterium rhizogenes infects a broad range of dicotyledonous hosts including legumes. A. rhizogenes-induced hairy roots are widely used as a tool to study gene function in plants. In legume plants in particular, the use of hairy roots has been a boon to work that F I G U R E 1 Sequence of hairy root induction in pea. (a) Germinated seeds ready for transformation; (b) seed coats removed; (c) radicle severed and coated with Agrobacterium rhizogenes containing ALSV infectious clones; (d) seedlings exhibiting hairy roots prior to transplanting into soil addresses symbiotic nitrogen fixation because these transgenic roots are able to be nodulated directly (Hansen et al., 1989). With advances in gene editing approaches, hairy roots have also been used to introduce CRISPR/Cas9 elements for genome modification in soybean (Cai et al., 2015), illustrating the efficacy of this technique for introducing recombinant DNA constructs.
Experiments with VIGS vectors are widely used for gene function studies because they are rapid and do not require generation of stable transformants. Several VIGS systems have been employed in legumes, including ALSV (Igarashi et al., 2009;Xiong et al., 2019). using the same protocol, we were not able to deliver ALSV to soybean and common bean. This is similar to previous attempts using an agroinjection inoculation method (Xiong et al., 2019).
Lentil plays an important role in alleviating malnutrition and micronutrient deficiencies in developing countries, providing an affordable source of dietary protein, carbohydrates, minerals, and fiber. A reference genome for pea (Kreplak et al., 2019) and a draft genome for lentil is available (Ramsay et al., 2019(Ramsay et al., , 2021 and genomic approaches have been applied to lentil breeding and identification of candidate genes (Kumar et al., 2021). The utility of gene silencing tools like VIGS have been identified (Kumar et al., 2015), but has seen limited success. Though not as robust as our success with pea, the ability to effect gene silencing in lentil is a promising step forward.
We expect that by using endogenous lentil sequences and with protocol refinement, this can develop into an effective tool for lentil gene silencing.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.