Targeted and random mutagenesis of cassava brown streak disease susceptibility factors reveal molecular determinants of disease severity

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.

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.

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.