Author Response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
It is well established that many potivirids (viruses in the Potiviridae family), particularly potyviruses (viruses in the Potyvirus genus), recruit (selectively) either eIF4E or eIF(iso)4E, while some others can use both of them to ensure a successful infection. CBSD caused by two potyvirids, i.e., ipomoviruses CBSV and UCBSV, severely impedes cassava production in West Africa. In a previous study (PBI, 2019), Gomez and Lin (co-first authors), et al. reported that cassava encodes five eIF4E proteins, including eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, nCBP-1 and nCBP-2, and CBSV VPg interacts with all of them (Co-IP data). Simultaneous CRISPR/Cas9-mediated editing of nCBp-1 and -2 in cassava significantly mitigates CBSD symptoms and incidence. In this study, Lin et al further generated all five eIF4E family single mutants as well as both eIF(iso)4E-1/-2 and nCBP-1/-2 double mutants in a farmer-preferred casava cultivar. They found that both eIF(iso)4E and nCBP double mutants show reduced symptom severity, and the latter is of better performance. Analysis of mutant sequences revealed one important point mutation, L51F of nCBP-,2 that may be essential for the interaction with VPg. The authors suggest that the introduction of the L51F mutation into all five eIF4E family proteins may lead to strong resistance. Overall I believe this is an important study enriching knowledge about eIF4E as a host factor/susceptibility factor of potyvirids and proposing new information for the development of high CBSD resistance in cassava. I suggest the following two major comments for authors to consider for improvement:
(1) As eIF(iso)4e-1/-2 or nCBP-1/-2 double mutants show resistance, why not try to generate a quadruple mutant? I believe it is technically possible through conventional breeding.
(2) I agree that L51F mutation may be important. But more evidence is needed to support this idea. For example, the authors may conduct a quantitative Y2H assay on the binding of VPg to each of the eIF4E (L51F) mutants. Such data may add as additional evidence to support your claim.
We thank the reviewer for their overall assessment. Regarding investigating a quadruple mutant, we agree that this is a logical next step to investigate. A conventional breeding approach with existing mutant lines, however, is problematic for several reasons; 1) cassava does not flower where this work was conducted, and 2) cassava is subject to inbreeding depression, resulting in both low seed set and considerable heterogeneity among progeny that do arise. Editing existing double mutants is possible, but would require a significant, multi-year investment to produce embryogenic tissue from existing lines and generate the new lines. Cassava has practical limits as a non-model plant. Given these constraints, we conclude that investigating a quadruple mutant is beyond the scope of the current work.
For investigating the HPL to HPF mutation in other cassava eIF4E-family proteins and their interaction with VPg in yeast, we have now completed this experiment and included the data in the paper. Notably we find that generating this mutant for eIF(iso)4E-2 attenuates VPg interaction without impairing eIF(iso)4E-2 accumulation, while similarly mutating nCBP-1 and eIF(iso)4E-1 results in total and reduced protein accumulation, respectively.
Reviewer #2 (Public review):
Summary:
The authors generated single and double knockout mutants for the eIF4E family members eIF4E, iso4E1, iso4E2, nCBP1, and nCBP2 in cassava. While a single knockout of these eIF4E genes did not abolish viral infection, the nCBP1/nCBP2 double knockout mutant displayed the weakest symptoms and viral infection. Through yeast two-hybrid screening, the nCBP-2 L51F mutant was identified, and the mutant was unable to interact with VPg, yet the nCBP-2 L51F mutant could complement the eIF4E yeast mutant. This L51F is a potentially important editing site for eIF4E.
Strengths:
This study systematically generated single and double knockout mutants for the eIF4E family members and investigated their antiviral activity. It also identified a L51F site as a potentially important antiviral editing site in eIF4E, however, its antiviral genetic evidence remains to be validated.
Weaknesses:
(1) The symptoms of the iso4E1 & iso4E2 double-knockout mutant are slightly alleviated, and those of the nCBP1 & nCBP2 double-knockout mutant are alleviated the most. If the iso4E1 & iso4E2 and nCBP1 & nCBP2 mutants are crossed to obtain quadruple-knockout mutant plants, whether the resistance of the quadruple mutant will be more excellent should be further investigated.
(2) Although the yeast two-hybrid identified the nCBP-2 L51F mutant, there is no direct biological evidence demonstrating its antiviral function. While the 6-amino acid deletion mutant (including L51F) showed attenuated symptoms, this deletion might be sufficient to cause loss-of-function of nCBP-2. These indirect observations cannot definitively establish that the L51F mutation specifically confers antiviral activity.
(3) Given that nCBP-2 can rescue yeast eIF4E mutants, introducing wild type and L51F nCBP2 into the Arabidopsis iso4e mutant viral infectious clones into yeast systems could clarify whether the L51F mutation (and the same mutations in eIF4E, iso4E1, iso4E2) abrogates their roles as viral susceptibility factors - critical genetic evidence currently missing.
We sincerely thank the reviewer for their constructive feedback.
With regards to investigating a quadruple eIF4E mutant, please see our response to reviewer 1.
The reviewer makes a salient point regarding the nCBP-2 L51F and K45_L51del mutations. Ideally, complementation of the ncbp double mutant with nCBP-2 L51F, followed by viral challenge, would address this question. However, the practical limitations, as noted in our response to reviewer 1, make this difficult within the context of this manuscript. We acknowledge that this is a limitation of our study and have been cautious in not overstating our conclusions.
Reviewer #3 (Public review):
In the manuscript, the authors generated several mutant plants defective in the eIF4E family proteins and detected cassava brown streak viruses (CBSVs) infection in these mutant plants. They found that CBSVs induced significantly lower disease scores and virus accumulation in the double mutant plants. Furthermore, they identified important conserved amino acid for the interaction between eIF4E protein and the VPg of CBSVs by yeast two hybrid screening. The experiments are well designed, however, some points need to be clarified:
(1) The authors reported that the ncbp1 ncbp2 double mutant plants were less sensitive to CBSVs infection in their previous study, and all the eIF4E family proteins interact with VPg. In order to identify the redundancy function of eIF4E family proteins, they generated mutants for all eIF4E family genes, however, these mutants are defective in different eIF4E genes, they did not generate multiple mutants (such as triple, quadruple mutants or else) except several double mutant plants, it is hard to identify the redundant function eIF4E family genes.
(2) The authors identified some key amino acids for the interaction between eIF4E and VPg such as the L51, it is interesting to complement ncbp1 ncbp2 double mutant plants with L51F form of eIF4E and double check the infection by CBSVs.
We thank the reviewer for their assessment and feedback.
Regarding analysis of higher-order mutants, please see our response to Reviewer #1’s public review.
For investigation of nCBP-2 L51F in planta, please see our response to Reviewer #2’s public review.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
(1) Since nCBP2 can complement a yeast mutant, it indicates that nCBP2 can also complement Arabidopsis. Wild-type nCBP2 should be introduced into the Arabidopsis iso4e mutant to determine whether it can complement Arabidopsis iso4e and whether the virus can re-establish the infection. The nCBP2 L51F mutant should also be introduced into the Arabidopsis iso4e mutant to see if this mutant fails to re-establish the virus infection. Similarly, eIF4E, iso4E1, iso4E2, nCBP1, etc., should be introduced into the Arabidopsis iso4e mutant to determine whether they can truly complement the virus-infected mutant Arabidopsis, while the L51F mutants cannot.
Arabidopsis encodes multiple eIF4E proteins, an nCBP protein, and an eIF(iso)4E protein, and knocking out the eIF(iso)4e gene specifically confers resistance to TuMV. Introducing cassava nCBP-2 into arabidopsis eif(iso)4e mutants is unlikely to restore TuMV susceptibility. Because TuMV belongs to a different genus than CBSV, we used the TuMV VPg interaction with arabidopsis eIF(iso)4E to test the generality of mutating the eIF4E HPL motif to HPF potyvirid VPg-eIF4E interaction. However, since this mutation disrupts arabidopsis eIF(iso)4E’s endogenous translation initiation activity in yeast, this mutant protein is not worth pursuing further. In contrast, cassava eIF(iso)4E-2 L27F retains translation initiation activity and has reduced interaction with CBSV VPg by quantitative yeast two-hybrid. It would be interesting to see if this particular mutant protein could interact with TuMV VPg, and if not, would then be worth testing for the ability to restore TuMV susceptibility in Arabidopsis eif(iso)4e. Unfortunately, we are unable to pursue these experiments at this time.
(2) Given that nCBP-2 can complement yeast eIF4E mutants, the authors may introduce viral infectious clones into yeast systems expressing nCBP-2 variants to determine whether nCBP-2 supports viral translation. This approach could further clarify whether the L51F mutation (and mutations in eIF4E, iso4E1, so4E2) abolishes their roles as viral susceptibility factors.
This is an intriguing suggestion, but challenging for a few reasons. First, an infectious clone of CBSV Naliendele isolate does not exist, although we have tried to construct one, without success. There is also no guarantee such a clone could infect yeast. We are aware of yeast being used as a surrogate host for a few plant viruses, such as Tomato bushy stunt virus and Brome mosaic virus but are unaware of a similar system for any potyvirid. Developing such a system would undoubtedly require a significant investmentbeyond the scope of this manuscript.
(3) Phenotypes of all mutant lines with and without virus inoculation in Table 1 should be presented.
Photos of un-challenged mutants are included in supplemental figures. Representative storage root symptoms for all lines have now been included in the supplemental figures as well.
(4) In Figure 1c, the results of viral accumulation assays should be presented for additional mutant lines beyond ncbp-1, ncbp-2, ncbp-1 nCBP-2 K45_L51del, and ncbp-1 ncbp-2, particularly eif(iso)4e-1 & eif(iso)4e-2#172 and eif(iso)4e-1 & eif(iso)4e-2#92.
We have previously found that subtle reductions in visible disease do not always translate to clear differences in viral titer when analyzed by qPCR (Gomez et al., 2018). As such, we focused on lines with the strongest phenotypes in viral titer experiments.
(5) Inconsistently, the ncbp-1 nCBP-2 K45_L51del line showed reduced symptoms compared to wild-type in Figures 1a and 1b, yet viral accumulation levels were comparable to wild-type in Figure 1c. The explanations for this discrepancy are required.
Please see our response to (4).
(6) Root phenotypic data for all mutant lines shown in Figure 1d should be presented.
Please see our response to (3).
(7) In Figure 2b, GST control pulldowns showed detectable proteins. This background signal requires explanation.
It is not uncommon to see weak signal in bead or tag-only negative control pulldown and IP reactions. Importantly, we see strong enrichment of VPg relative to these controls in our experimental samples.
(8) Contrary to the abstract's implication, Figure 5c indicates that the L51F mutation impacts yeast growth, suggesting potential pleiotropic effects of this mutant.
We interpret the results to be that nCBP2 L51F does not fully complement the yeast eif4e mutation, rather than nCBP2 L51F impacts yeast growth.
(9) In vivo protein-protein interaction assays (e.g., co-immunoprecipitation) should be performed to complement the in vitro GST pull-down data in Figure 6.
We appreciate the desire for these experiments and agree that they would bolster our Y2H and pulldown data. Unfortunately, we are not able to complete these experiments at this time, so have been careful not to over interpret the data.
(10) Since the AteIF(iso)4E L28F mutant fails to complement yeast, the authors should test whether introducing the L51F mutation into other family members (eIF4E, iso4E1, iso4E2, nCBP1) preserves their yeast complementation capacity.
This has now been done for additional cassava eIF4E-family proteins.
(11) Indicate molecular weight sizes in all Western blots.
This was done. As differences in buffer formulations between gel types can affect the mobility and thus apparent molecular weight of markers, we have provided in the methods section SDS-PAGE gel chemistries and specific protein ladders used in this study. Importantly we note in our experience that certain markers, in relation to proteins of interest, can vary up to 15 kDa between gel chemistries.
(12) Figures 4d,e are not provided in the paper. Based on the content of the paper, the description in the paper likely corresponds to Figures 5c, d.
Thank you for catching this error, this has now been corrected.
