TME419 CRISPR mutant genotypes

Endpoint CBSD storage root disease severity in cassava eIF4E-family mutants

a, b) Endpoint aerial disease (a) and storage root necrosis (b) observed in cassava eIF4E-family single and double mutants after challenge with CBSV isolate Naliendele. Data presented are an aggregate from all experimental replicates. Leaf and stem disease symptoms are evaluated separately on a 0-5 scale and then added together for a total aerial symptom score. Storage root necrosis severity was scored on a 1 to 5 scale, 1 being asymptomatic and 5 corresponding to severe necrosis throughout all storage roots of a plant. Mann-Whitney U test was used to detect statistical differences with an alpha = 0.05. *≤ 0.05, ** ≤ 0.01, *** ≤ 0.001. Asterisks above boxplots denote differences from wild type, while asterisks above brackets indicate differences between indicated genotypes. c) qRT-PCR quantification of storage root CBSV viral levels from challenged mutants. CBSV HAM1 was quantified relative to cassava PP2A to obtain HAM1 normalized relative quantities (NRQs). HAM1 NRQs for each genotype are presented as relative to the mean of wild type. Statistical differences were detected with the Mann-Whitney U test as described in (a, b). d) Representative storage root sections from wild type and ncbp-1 ncbp-2 #213 plants challenged with CBSV. Scale bar denotes 1 cm.

Interaction of GST-cassava eIF4E family proteins and CBSV VPg in vitro

a, b) GST-pulldown assays were performed with CBSV VPg-6xHis-3xFLAG and GST or GST-tagged cassava eIF4E-family proteins. Proteins were expressed in and purified from E. coli. Human (Hs) eIF4E was also tested in (a). c) Model for eIF4E-mediated resistance against potyvirids. Cartoon depicts potyvirid genome-linked VPg protein bound to eIF4E-family protein in order to cause disease, while mutations that disrupt this interaction results in resistance. d) Coimmunoprecipitation of YFP or YFP-eIF4E-family proteins with TuMV 6xHis-VPg-6xHIS-3xFLAG. VPg was expressed in and purified from E. coli and then mixed with extract from N. benthamiana leaves expressing YFP or YFP-fusion proteins. e) Arabidopsis eIF(iso)4E residues 92 through 150 aligned with homologous regions of cassava eIF4E-family proteins. W92 and K150, marked red, are necessary for interaction with TuMV VPg. * indicates conservation across all aligned proteins,: indicates conservation of amino acids with strongly similar properties, and. indicates conservation of amino acids with weakly similar properties. f) GST-pulldown assay as in a) and b) with CBSV VPg incubated with either GST, GST-nCBP-2, and GST-nCBP-2W118L/N170E. g) Comparison of nCBP-2 and nCBP-2W118L/N170E interaction with CBSV VPg by quantitative yeast two-hybrid assay. h) Western blot analysis of nCBP-2 and VPg expression in yeast from g).

A yeast two-hybrid loss-of-affinity screen identifies nCBP-2 residues necessary for VPg interaction

a) Workflow for using a yeast two-hybrid approach to identify nCBP-2 mutants that lose affinity for CBSV Naliendele VPg. The resultant pEG202 prey vector encodes nCBP-2 fused to the LexA DNA binding domain. pJG4-5 encodes CBSV VPg fused to the B42 transcriptional activation domain. The EGY48 yeast strain used harbors the pSH18-34 reporter plasmid containing lacZ driven by the LexA operator. b) Ventral and dorsal views of cassava nCBP-2 structure produced using the Phyre2 server (Kelley et al., 2015). nCBP-2 structure of amino acids 49 through 226 was predicted using wheat eIF4E (PDB ID: 2IDR) as a template. Mutated amino acid residues in nCBP-2 mutants that lose VPg affinity and either rescue or fail to rescue eif4e yeast are highlighted in green and pink, respectively. W118 and N170, homologous with TuMV VPg interacting residues of Arabidopsis eIF(iso)4E, are highlighted in blue.

Summary of nCBP-2 mutants found to lose affinity for CBSV VPg

T93C eif4e yeast complementation by nCBP-2 mutants

a) Schematic for testing translation initiation activity of nCBP-2 variants expressed from the pG1.1 plasmid in T93C eif4e conditional mutant yeast. b) Left panels: T93C yeast transformed with pG1.1 empty vector or pG1.1 encoding various eIF4E-proteins grown on SD dropout media supplemented with galactose allowing for conditional expression of yeast eIF4E from the pGAL1-eIF4E plasmid. Right panels: same yeast strains from left panel grown on dropout media supplemented with glucose, preventing complementation by the pGAL1-eIF4E plasmid. c) Western blot analysis of 3xFLAG tagged nCBP-2 variant expression in yeast strains used in a). Coomassie stained membrane is presented for assessing loading control.

Effects of HPL motif mutation on nCBP-2 affinity for CBSV VPg and translation initiation

a) Alignment of human eIF4E protein sequence with variants of cassava nCBP-2. The conserved HPL motif is boxed in red and the L51F mutation in nCBP-2 is highlighted in cyan. Asterisks denote conserved amino acid residues. b) Quantitative yeast two-hybrid analysis of CBSV VPg interaction with nCBP-2 variants. c) T93C eif4e yeast transformed with pG1.1 encoding Arabidopsis eIF4E1 or cassava nCBP-2 variants under the control of a constitutive promoter. nCBP-2 variants are N-terminally tagged with 3xFLAG. The T93C yeast strain harbors a plasmid encoding galactose-inducible and glucose-repressible yeast eIF4E. d) Western blot analysis of nCBP-2 expression in yeast used in c). A segment of coomassie stained membrane is presented for loading control.

nCBP-2K45_L51del and nCBP-2L51F exhibit reduced affinity for CBSV VPg, in vitro

a) GST pulldown experiment assessing the ability of GST-nCBP-2 variants to bind CBSV VPg. Full-length protein eluate band intensities are presented underneath the eluate western blots and are expressed relative to the sample with the strongest band intensity. b) Analysis of eluate VPg band intensity across three GST-pulldown experiments. Band intensities are normalized to account for differing amounts of GST-fusion protein pulled down and expressed relative to VPg intensity in the GST-nCBP-2 sample.

Effects of mutating the HPL motif leucine in Arabidopsis eIF(iso)4E

a) Quantitative yeast two-hybrid analysis of TuMV VPg interaction with wild type Arabidopsis eIF(iso)4E alongside W92L/K149E and L28F mutants. b) T93C eif4e yeast complementation assay with wild-type and mutant eIF4E-family genes from Arabidopsis and cassava. c) Western blot analysis of eIF(iso)4E and nCBP-2 variants from cells used in (b).