A cassava protoplast system for screening genes associated with the response to South African cassava mosaic virus

Two genomic gRNA targets, gRNA1 (forward strand: GCGCAGATTCAAGCACTCGA) and gRNA2 (reverse strand: ACGTCCATTGGCGATGATAG),were identified using the CRISPOR version 4.7 web-based program (www.crispor.tefor.net; [29]) and used in designing a duplex sgRNA that included an Arabidopsis thaliana U6-26 promoter, the sgRNA scaffold and terminator for each gRNA sequence. Synthesis of the duplex sgRNA was outsourced to Inqaba Biotec (Pretoria, South Africa) and the duplex was cloned into the pCambia1380 vector. A Cas9 insert (Tobacco mosaic virus promoter + Cas9 gene + eGFP gene + Hsp terminator) from the pl1m-f2-p35s-cas9-egfp-nucleo-thsp Golden Gate vector was cloned into the pCambia1380-gRNA construct. The construct was confirmed by restriction digestion and sequencing.

Sterile nodal cultures of cassava (cv.60444, T200 and TME3 genotypes) were grown for 4 weeks at 28 °C (3,000 lx; 12/12 h light/darkness) on ½ Murashige and Skoog (MS) medium (2.2 g Murashige and Skoog Basal Medium, 2% sucrose, 0.002 mM CuSO₄, 0.78% plant tissue culture agar) [67] to provide 3 independent biological replicates of each genotype. For each treatment, 0.3 g of fully expanded leaves were transversely sliced into 2–3 mm strips, which were plasmolysed by immersion in CPW9M medium (0.5 M mannitol, 27.2 mg KH₂PO₄, 100 mg KNO₃, 150 mg CaCl₂, 250 mg MgSO₄, 2.5 mg Fe₂(SO₄)₃.6H₂0, 0.6 mg KI, 0.00025 mg CuSO₄ per litre; pH 5.8) for 1 h [8]. The strips were vacuum-infiltrated in enzyme digestion solution (5 mM morpholinoethanesulphonic acid (MES), 1.6% cellulase, 0.8% macerozyme, CPW9M medium) for 30 min and then incubated at 25 °C in the dark at 40 rpm for 16 h. Protoplasts were released by shaking the digested tissue at 80 rpm for 5 min and purified by filtering through a 75 μm sieve. The filtrate was centrifuged at 100 g and the protoplast pellet washed twice in CPW9M medium. Protoplast integrity was checked using the Olympus BX 63 OM/FM microscope (Olympus Scientific Solutions, Massachusetts, USA), viability was determined by Evans’ Blue Dye staining [33], and quantification conducted via flow cytometry using the BD Accuri™ C6 flow cytometer (BD Biosciences, New Jersey, USA). Flow cytometric data were analysed using FCS Express 7 Research Edition software (Treestar, Inc, Oregon, USA). Protoplasts were resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl₂, 4 mM MES; pH 5.8) [96]) to a concentration of 10⁴ cells per ml.

For each of the 3 biological replicates for each cassava genotype, 15 μg of the CRISPR construct and 4 μg each of pBIN19-SACMV-DNA-A and pBIN19-SACMV-DNA-B infectious clones [12] were mixed with 1 mL of protoplasts and 25% polyethylene glycol 4000 (PEG4000), and incubated at room temperature for 20 min. The mixture was gently diluted with 3 volumes of W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.8) and centrifuged twice at 100 g for 2 min. Protoplasts were resuspended in 300 μL of WI solution (4 mM MES, 0.5 M mannitol, 20 mM KCl, pH 5.8) [98] and incubated overnight in the dark at room temperature to induce gene expression. The expression of eGFP was checked using fluorescence microscopy to confirm protoplast transformation. Protoplasts were washed with CPW9M medium at 24 h post-transfection.

DNA was extracted from the transformed protoplasts 24 h post-transfection (hpt) using QIAzol Lysis Reagent according to a user-developed protocol (www.qiagen.com/it/resources/; Qiagen, Maryland, USA). Quantitative PCR (qPCR) for SACMV relative viral load quantitation using DpnI-digested (ThermoFisher Scientific, Massachusetts, USA) DNA as template was performed in triplicate using forward (5` GGCTAGTTCCCGGATTACAT 3`) and reverse (5` GACAAGGACGGAGACACC 3`) primers, and 18S rRNA as the reference gene. The exonic region of MeE3L was amplified using the Phusion U Green Hot Start DNA Polymerase (ThermoFisher Scientific) with forward (5` CGCGCAGATTCAAGC 3`) and reverse (5` TGTCCACATGGAATGAAAG 3`) primers. Sequencing of amplicons on the ABI 3500XL Genetic Analyzer was outsourced to Inqaba Biotec (Pretoria, South Africa), and sequencing results were analysed for variation and alignment using TIDE web tool (https://tide.deskgen.com/; [16]) and MAFTT version 7 (https://mafft.cbrc.jp/alignment/software/, [79]) before being employed as query terms for protein structure and binding prediction using the I-TASSER On-line Server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/, [97]) and protein similarity search in the Protein Data Bank [10]. Analysed sequences were used to construct a phylogenetic tree using the MEGA X software based on the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model [40]. Frequency of clones with altered sequence was obtained by expressing a number of amplicons from a polyclonal mix with sequence alteration as a ratio of 10 amplicons sequenced. Mutations were determined by aligning amplicon sequences with wild-type reference AM560-2 [14] and TME3 (RefSeq ID: RSFT01000007, GenBank assembly GCA_003957995.1 (unpublished data)) MeE3L homologs. Alignment was conducted on MEGA-X [40] using the CLUSTAL W algorithm for multiple sequence alignment [44].

RNA was extracted from the transformed protoplasts 24 hpt using QIAzol Lysis Reagent and according to the manufacturer’s (Qiagen, Maryland, USA) protocol. First strand cDNA synthesis using RNA as template was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Massachusetts, USA). Reverse transcriptase qPCR for MeE3L relative expression quantitation was performed in triplicate with cDNA as template using the Maxima SYBR Green/ROX qPCR Master Mix (2X) (ThermoFisher Scientific, Massachusetts, USA) with forward (5` CGCGCAGATTCAAGC 3`) and reverse (5` TGTCCACATGGAATGAAAG 3`) primers according to the manufacturer’s protocol using 18S rRNA as the reference gene. The qPCR data was analysed by one-way ANOVA and Tukey’s HSD test (p < 0.05). Relative expression and relative viral load of MeE3L and SACMV, respectively, were determined using the 2^−ΔΔCT method [54].

The in vitro growth of the cassava plantlets was conducted under controlled, sterile conditions and only young, expanded leaves were used as donors (Fig. 1a). Different sizes (~ 15–35 µm) of cassava protoplasts of round and irregular shape were observed (Fig. 1b–d). The majority of protoplasts were round with chloroplasts positioned around the perimeter of a central vacuole. The viability of protoplasts was at least 85% as shown by staining with Evans’ Blue Dye (Fig. 2a–c). Protoplast yields were 4.90–6.36 × 10⁶/g fresh weight (FW) (Additional File 1). The integrity of isolated protoplasts was analysed by flow cytometry forward versus side scatter (FSC vs SSC) gating [32] prior to transfection (Fig. 2d–f). The presence of protoplasts in the gated area as well as irregularly-shaped debris outside the gated area was detected. Approximately 10⁴ protoplasts of each cassava genotype were transfected with 15 µg eGFP-tagged CRISPR construct and/or 4 µg SACMV infectious clones using PEG-mediated transformation. Stability of transient expression was verified by detection of eGFP expression 24 hpt, showing that at least 90% of protoplasts had been successfully transformed (Fig. 2g, h).

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PCR amplification of the MeE3L partial transcript from leaves revealed that the susceptible T200 landrace homolog nucleotide sequence is slightly longer than the tolerant TME3 homolog (Fig. 3b). Sequencing of the exonic region of susceptible T200 MeE3L showed that this is due to a 53 bp insertion mutation of 9 TGAGAA nucleotide repeats that are absent in model cv.60444 and tolerant TME3 (Fig. 3a). The resulting frameshift introduces a stop codon corresponding to amino acid 141 (Fig. 3c, d). Computational analysis revealed that the truncated susceptible T200 MeE3L homolog is structurally distinct from the reference genome (AM560-2), model cv.60444 and tolerant TME3 MeE3L homologs (Fig. 3e). It is also not significantly similar in structure to any protein in the Protein Data Bank [10], and it is not confidently predicted to bind any ligands. The other three (AM560-2, cv.60444 and TME3) homologs are structurally closest to the cIAP1 inhibitor of apoptosis protein which contains a RING domain with E3 ligase activity for autoubiquitination and modulates cell death [25]. An analysis of the phylogeny of E3 ligase homologs in plants (Fig. 3f) reveals that although the MeE3Ls share Hevea brasiliensis as a common ancestor, the susceptible T200 homolog is significantly more evolutionarily distant from AM560-2 than the TME3 and cv.60444 homologs.

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Tukey’s HSD test showed significant differences in the qPCR data derived from SACMV-, CRISPR- and SACMV + CRISPR-transformed protoplasts (p < 0.05) (Additional File 2). Relative SACMV DNA accumulation in wild-type protoplasts was highest in susceptible T200 at eightfold, compared to threefold relative to the 18S rRNA gene in model cv.60444 and tolerant TME3 at 24 hpt. There was a substantial increase in accumulation of SACMV DNA in model cv.60444, susceptible T200 and tolerant TME3 (12-fold and fourfold respectively) in CRISPR-transformed protoplasts (Fig. 4a). MeE3L expression was significantly upregulated with a log fold change of 1 and 9 in susceptible T200 and tolerant TME3 SACMV-infected protoplasts relative to MeE3L expression in untransformed protoplasts, respectively. There was significant upregulated expression of MeE3L in tolerant TME3 but no significant effect on susceptible T200 MeE3L expression in CRISPR-transformed protoplasts. No significant change in MeE3L expression was observed under all conditions in model cv.60444 (Fig. 4b).

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Predicted MeE3L protein primary structures based on the MeE3L genomic sequence in SACMV-infected protoplasts showed stop mutations (amino acid residues 133 and 99 respectively) (Fig. 4c, d) upstream of the RING domain in susceptible T200 and tolerant TME3 variants, compared to uninfected controls and reference sequence AM560-2. Sequencing of MeE3L from SACMV-infected tolerant TME3 protoplasts revealed multiple random single base mutations along the length of MeE3L which translate to substitutions by altered amino acids (Fig. 4e). CRISPR editing efficiency was determined based on Sanger sequencing of 10 amplicons from a polyclonal mix. Sequencing indicated 80% and 50% mutation frequency for the gRNA1 and gRNA2 targets respectively. All mutations were substitution mutations and translated to 20% and 50% mutation efficiency for the gRNA1 and gRNA2 targets respectively in the predicted protein sequence (Fig. 4f). The turnaround time from commencement of plant tissue culture to gene expression assaying was 7 weeks (Fig. 4g).

Protoplasts were chosen for this transient gene expression study because they can conveniently and efficiently be transformed with several DNA constructs simultaneously, and they allow higher resolution imaging compared to cells in intact tissue [27]. Additionally, they can be used for high-throughput efficient screening of candidate genes, and those genes that show an effect can then be silenced by virus induced gene silencing (VIGS) in planta, which takes considerably longer (3–4 months) and requires more complicated procedures for the non-model host cassava. Leaf mesophyll was used as the source of protoplasts because SACMV exerts its effects mainly in the leaves, where symptoms arise. Leaf mesophyll protoplasts therefore provide functional information [27] relating to the effect of SACMV at leaf tissue level. Round and irregularly-shaped protoplasts of different sizes were observed (Fig. 1b–d), although spherical leaf mesophyll protoplasts dominated, as generally reported [96].

A previously determined enzyme concentration (1.6% cellulase, 0.8% macerozyme) that is suitable for obtaining the optimum number of viable protoplasts was used for leaf cell wall digestion [96]. The viability of protoplasts in this study was at least 85% although cassava protoplast viability of up to 95% has been reported (International Plant Research Institute, 1984). The long digestion period (16 h) was ruled out as the cause of death for ~ 15% of protoplasts as this reportedly does not induce serious damage in protoplasts [87]. It has been reported that micro-propagated plants grown in vitro lack epicuticular wax and thus allow rapid enzyme penetration [40]. Cassava, however, has a thick epicuticular layer which necessitates the long digestion period of 16 h compared to 0.3-1 h for Arabidopsis [95]. Macerating enzymes such as macerase are known to cause wound reactions in protoplasts because of their degradation of the cell wall, which may lead to necrosis [34], while cellulase is known to exert inadequate enzymatic activity at low concentrations and higher concentrations have no benefit or detriment [88]. Therefore, a balance between digestion enzyme concentration and viability is essential in order to obtain the optimum yield of viable good quality protoplasts. Cassava protoplast viability presented herein may differ from previously reported percentages possibly due to differences in the cassava genotypes used, and in this particular case could be due to the particular physiological characteristics of the African cassava landraces from which protoplasts were derived.

Although lower than the previously reported yields of 4.4 × 10⁷ protoplasts/g FW leaves from M. esculenta cv. South China 8 [96] and 1.9 × 10⁷ protoplasts/g FW leaves from M. esculenta cv. M. Thai 8 [8], the protoplast yields in this present study (4.90–6.36 × 10⁶/g FW) were sufficient to provide the recommended number of protoplasts (10⁴–10⁷) required for each transfection [98]. Both pre-treatment of leaves in the dark (24–72 h) [84] and vacuum infiltration before enzyme digestion [68] have been shown to intensify enzyme penetration in bean leaves. We found that pre-treatment resulted in release of much undesirable plant debris alongside protoplasts, and that vacuum infiltration did indeed help increase protoplast yield. Flow cytometry indicated a high concentration of protoplasts compared to irregularly-shaped debris. Based on microscopy images of purified protoplasts (Fig. 1b–d), the irregular debris outside the flow cytometry gated area was deemed to be free chloroplasts, plasmolysed cells, undigested cell wall fragments and other aggregates arising from the long digestion period of leaf material.

Stability of protoplast transformation was confirmed by expression of eGFP from the CRISPR construct at 24 hpt following PEG-mediated transfection of about 10⁴ protoplasts with 15 µg CRISPR construct and 4 µg SACMV infectious clones. Protoplasts were deemed unsuitable for further analysis from 36 hpt as they rapidly lost viability. PEG-mediated plant protoplast transfection with plasmid DNA is a well-established procedure (Hayashimoto et al. 1990; [55]) and a popular protocol uses 10 μg DNA to transfect 2 × 10⁴ Arabidopsis protoplasts [98]. At least 5 µg of plasmid DNA have previously been used to transform 10⁶ tobacco protoplasts with African cassava mosaic virus (ACMV) (Ermak et al. 1993) and Cowpea mosaic comovirus [93]. Highly efficient co-expression of multiple constructs in plant protoplasts has been reported [18, 91] and virus infectious clones have also been used in conjunction with other plasmid constructs for co-inoculation of plant protoplasts [20]. Nicotiana tabacum protoplasts have been co-transformed with 5 µg eGFP construct, 3 μg siRNA and 4 μg each of ACMV or East African cassava mosaic virus (EACMV) with DNA/RNA extraction at 36 and 48 hpt [89]. To our knowledge, the present study is the first report of co-transformation of cassava protoplasts with a CRISPR construct and geminivirus infectious clones. The number of constructs used for transformation can be increased for high-throughput studies to target multiple genes simultaneously since it has been shown that transformation efficiency is independent of plasmid amount [55].

The percentage mutation frequency of 50–80% at targeted sites in the cassava genome is comparable to the 70% and 60% attained in cassava [96] and N. tabacum [52] protoplasts respectively, and considerably less than the 100% previously obtained in cassava plants [70]. CRISPR targets with GC content greater than 50% are known to achieve higher efficiency than those with less than 50% [59], and this may explain why gRNA1 target sequence (55% GC content) has considerably higher mutation frequency than gRNA2 target sequence (50% GC content).

The frameshift resulting from the insertion mutation in susceptible T200 MeE3L introduces a stop codon upstream of the RING domain, thus encoding a truncated protein in which the C3HC4-type RING finger motif is absent. Essentially, the E3 ligase domain in the T200 MeE3L would not be translated because of this mutation, making its potential protein product non-functional with respect to this E3 ligase activity. We were able to amplify the T200 MeE3L exon from cDNA, showing that this gene is transcribed, but we have no evidence of its translation or lack thereof. It is proposed that the loss of this substantial portion of the T200 MeE3L C-terminal region would not only avert E3 ligase activity but may also alter the spatial chemical conformation necessary for any other interactions (such as binding the ubiquitin-conjugated E2 and substrate) to occur. The protocol in this present study enables gene sequence comparison among wild type cassava genotypes, to determine the presence of single nucleotide polymorphisms (SNPs) or other forms of mutations, and to form a basis for in planta exploration of phenotypic differences among cassava genotypes.

The AM560-2, TME3, and cv.60444 cassava genotypes have previously been shown to cluster together under their nearest ancestor, Hevea brasiliensis [14]. The evolutionary history of T200, a southern African landrace, is unknown and phylogenetic analysis suggests that the T200 MeE3L evolved after the cv.60444 and TME3 variants. It is known that wild plants in natural ecosystems co-evolve with their virus partners. While it is recognized that there is a relationship between virus virulence/pathogenicity and co-adaptation to plant hosts (Sacristan and Garcia-Arenal 2008), information regarding how viruses apply selective pressure to alter plant susceptibility is not known. A study of Drosophila and its host-specific viruses found that coevolution may cause sustained genetic variation in susceptibility [26]. This may explain why a southern African cassava landrace is highly susceptible to SACMV that appears to have migrated south from its origin, suspected to be in east Africa or the south-west Indian Ocean islands such as Madagascar [24] that are geographically separated from the African continent [45]. South African cassava mosaic virus is a recombinant between East African cassava mosaic virus and two other unknown geminiviruses which contributed the AC4 and IR regions [12], and moved southwards into Mozambique, Zimbabwe and South Africa where it may have encountered the T200 landrace. Subsequent to its first discovery in South Africa, SACMV has been reported in Zimbabwe [15] and Madagascar [24]. It is known that infection with a new recombinant begomovirus requires the host to adjust to minor or major differences in virus-host interactions [64]. It is suggested that the T200 landrace and SACMV may still be in the process of co-adaption, which would explain why T200 exhibits extreme susceptibility to SACMV. We speculate that the MeE3L is either a paralog in T200 or it was introgressed from a wild relative in southern Africa.

Quantitative PCR is a well-established method for precise quantitation of viral DNA amount in infected tissue and it requires a host reference gene with stable expression patterns under experimental conditions as the internal control for correct data normalisation [65]. Data from the qPCR measurement of SACMV DNA accumulation (relative to the 18S rRNA gene) show that SACMV DNA accumulates in cassava protoplasts, correlating well with previous reports of geminivirus DNA accumulation in planta and in vitro. Quantitative detection of African cassava mosaic virus and East African cassava mosaic virus using qPCR has been reported [72] and SACMV titre, in particular, has been assayed in planta in Arabidopsis [74] and cassava [5]. Replication of the geminivirus, Cassava brown streak virus, in cassava leaf mesophyll protoplasts has been assayed at 6 hpt [7] and it has been reported that there was significant viral DNA accumulation in tobacco BY-2 protoplasts 36 and 48 hpt by co-inoculating with infectious ACMV and EACMV clones and siRNA [89]. The present study is the first to report accumulation of SACMV DNA in cassava protoplasts.

Based on previously reported in planta evidence [5] and the known presence of a CMD2 locus in tolerant TME3 [2], it was expected that SACMV DNA accumulation would be genotype-dependent and significantly lower in TME3 than in the model cv.60444 and susceptible T200 protoplasts. Interestingly, there was differential SACMV accumulation in CRISPR-transformed cassava protoplasts expressing the gene-edited MeE3L. The upregulation of SACMV DNA accumulation in susceptible T200 and tolerant TME3 in the presence of mutant MeE3L suggests a role for MeE3L as one of the host genes involved in the response to SACMV infection. CRISPR-associated modification of MeE3L may enhance SACMV DNA accumulation in susceptible T200 and tolerant TME3 by interfering with the ubiquitin proteasome system-dependent tolerance/resistance response mechanisms of cassava.

Geminiviruses elude plant defense mechanisms by hijacking and redirecting ubiquitination, and interfering with responses regulated by ubiquitin E3 ligases (including responses to jasmonates, auxins, gibberellins, ethylene, abscisic acid) [58]. It follows then that alterations to E3 ligase genomic sequences may alter E3 ligase expression patterns during viral infection, as viruses are known to modulate RNA levels to enhance infection [90]. Both plant viruses and CRISPR systems are known to induce mutations in the genome [22, 60, 82], and the employment of both against the MeE3L would provide an indication whether MeE3L may be involved in the plant’s response to SACMV.

Previously, plant E3 ligases have been shown to be induced by viral infection [19, 23, 43] and plant defence elicitors [51, 80]. It is known that geminiviruses interact with plant E3 ligases and induce their up- or down-regulation to promote infection or undergo degradation [43, 58, 86]. Results presented herein indicate that in TME3 protoplasts, MeE3L expression is upregulated during SACMV infection. The concurrent CRISPR-mediated gene editing of MeE3L and infection with SACMV appears to induce increased expression of the MeE3L, suggesting that MeE3L’s specific base sequence is important for the interaction between the virus and the plant host. The muted response of the T200 MeE3L to all treatments was expected given its nonsense mutation which silences the RING domain responsible for E3 ligase activity. However, the muted response of model cv.60444 MeE3L was unexpected and suggests that this MeE3L sequence variant is not responsive to SACMV infection.

The MeE3L homolog sequences in SACMV-infected protoplasts reflect a silenced RING domain, suggesting that SACMV may possibly induce silencing of the RING domain in order to achieve full infection of the host. The concomitant increase in SACMV DNA accumulation and gene-edited MeE3L in TME3 points to the response of MeE3L to SACMV being more directed at advancing susceptibility. There is evidence for geminiviral (Tomato yellow leaf curl sardinia virus) silencing of a plant E3 ligase, RHF2A, to promote infection [58] and impairment of plant defence during Cabbage leaf curl virus (CaLCuV) infection due to inhibition of a RING E3 ligase [81]. The present study provides further evidence that geminiviruses may interfere with activity of plant E3 ligases.

Functions of E3 ligases in regulating immunity systems are orchestrated at the interface of host-virus interactions [100] and some of these interactions occur in the nucleus [42]. Sequencing of genomic MeE3L from SACMV-infected TME3 protoplasts revealed multiple random single base mutations along the length of MeE3L, which translate to amino acid substitution (Fig. 4e). While these mutations do not alter the reading frame, they are predicted to silence the whole protein and not just the RING domain. The resulting disordered protein would presumably not only lack RING E3 ligase activity, but also the E2 and substrate binding activity. These mutations were present and similar in all 10 genomic DNA amplicons derived from the polyclonal mixes of each of 3 biological replicates. Similar mutations encoding multiple stop codons have been observed in an Argonaute 4-encoding gene (Manes.18g121900) from SACMV-transformed tolerant TME3 protoplasts (unpublished data; Chatukuta and Rey), indicating that other host genes may be similarly affected by SACMV infection.

Interestingly, the discovery of mutations in genomic DNA presented herein possibly point to a yet unknown geminivirus-induced host mechanism for genome editing. Geminiviruses are known to induce the expression of genes related to repair of double-stranded breaks (DSBs) and DNA synthesis [57], and to promote somatic homologous recombination [78]. Some E3 ligases and viral proteins can localise to the nucleus, such as the tobacco E3 ligase, NtHUB1 which has a nuclear localisation sequence, is recruited by geminiviral Rep protein, and co-localises and interacts with the Rep protein to monoubiquitinate cellular chromatin and thus enable infection [42]. The viral coat protein, CP, also has a nuclear localisation signal, can localise in the nucleolus and nucleoplasm, and facilitates entry of ssDNA into the nucleus [39, 92]. However, the mechanisms for SACMV-mediated gene mutation induction in cassava protoplasts are yet to be investigated.

Ubiquitin ligases are abundant in plants and provide substrate specificity to target particular proteins. In Arabidopsis alone, RING E3 ligases make up 499 out of over 1,500 E3 ligases [62]. A comparison of E3 ligase and E3 ligase complex-associated gene expression during other plant geminivirus infection studies (Additional File 3) was conducted to determine whether MeE3L’s response to SACMV is geminivirus-specific or host-dependent.

In susceptible cassava, E3 ligase expression is downregulated during SACMV infection at early, middle and late time points (12, 32 and 64 days post infection (dpi)) but there is no differential expression of E3 ligases in tolerant cassava at any time point [5]. However, no differential expression of E3 ligases is recorded during SACMV infection of Arabidopsis which is susceptible (Pierce and Rey 2013). A study of transcriptomic responses to geminivirus Tomato leaf curl New Delhi virus (ToLCNDV) infection in potato found that five E3 ligases in the susceptible cultivar and two in the tolerant cultivar are upregulated at 30 dpi [35] while the geminivirus Tomato yellow leaf curl virus (TYLCSV) has been shown to induce upregulation of E3 ligases in susceptible tomato at 42 dpi, except in the case of a CUL1 which is downregulated [63]. A transcriptome study of Arabidopsis during geminivirus CaLCuV infection found that, out of 1570 E3 ligases, 149 were up-regulated and 23 were downregulated [9]. The CaLCuV AC2 protein, in particular, induces downregulation of two E3 ligases in Arabidopsis [53]. These findings, together with the current study, prove that plant E3 ligase responses to geminivirus infection are neither uniform nor similar, but they vary according to the specific geminivirus and host involved in the interaction.

Responses of cassava to the ssRNA potyviruses Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) with respect to E3 ligase expression variably show both downregulation and upregulation in the susceptible varieties. Interestingly, there is no differential expression of E3 ligases in resistant cassava varieties except in Kaleso where a CUL1 is upregulated and a RZPF34 is downregulated [6, 7, 61]. This variable expression of E3 ligases with respect to the virus in the same host suggests that while responses to viral infection are host-dependent, they are also modulated according to the particular virus infecting the plant.

SACMV infection in planta is associated with occurrence of severe symptoms leading to persistent severe infection in susceptible T200 and mild symptoms with recovery at 67 days post infection (dpi) in tolerant TME3 [5]. The SACMV-induced genetic mutations and differential expression of MeE3L post-infection in TME3 and T200 indicate that it is one of the genes involved in the plant’s response to the virus. In planta proteome data from our laboratory shows that during SACMV infection, an E3 ligase (Manes.08G075100) is upregulated in susceptible T200 and downregulated in tolerant TME3 cassava plants at 32 and 67 dpi (unpublished data; Rey), supporting indications from the protoplast system that E3 ligases are responsive to SACMV infection.

This protocol presented herein suffers some limitations due to the independent cell nature of protoplasts and the short-lived viability of cassava protoplasts in particular. It cannot be used to study cell wall-related genes, cell-to-cell signalling, intercellular movement, long-term responses, or long-term stability of CRISPR-induced gene edits. Further, the use of mesophyll protoplasts may not be suitable to correlate responses in other organs such as roots or flowers. The widely used T7 endonuclease I (T7EI) assay for detecting gene editing activity produced inconclusive results for this present study, and therefore gene editing was indicated by restriction digestion and confirmed by sequencing. It has been reported that CRISPR-Cas9 activity is more accurately reflected by Next Generation Sequencing (NGS) [83]. In planta validation of results from this protoplast system protocol, particularly overexpression and virus-induced gene silencing (VIGS) of targeted genes, as well as functional and interaction studies, must be conducted to confirm the specific roles played by candidate genes in the host-virus interaction.