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. 2024 May 28:15:1404160.
doi: 10.3389/fpls.2024.1404160. eCollection 2024.

Exploring the source of TYLCV resistance in Nicotiana benthamiana

Satomi Hayashi  1   2 , Jacqueline M Souvan  1 , Julia Bally  1   2 , Felipe F de Felippes  1 , Peter M Waterhouse  1   2
Affiliations

Exploring the source of TYLCV resistance in Nicotiana benthamiana

Satomi Hayashi et al. Front Plant Sci. .

Abstract

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes.

Keywords: Nicotiana benthamiana; RNA-dependent RNA polymerase; Ty genes; disease resistance; tomato yellow leaf curl virus.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
N. benthamiana LAB and QLD accessions displaying differences in susceptibility to TYLCV. (A) LAB and QLD plants at 3 weeks post Agroinfiltration with the Agrobacterium either carrying the TYLCV infectious clone (TYLCV), or no-vector control (Mock); (B) Development of chlorotic leaf patch at the site of infiltration after 5 dpi; (C) Copy number of viral genome detected by qPCR in the systemic tissue at 3 wpi (n=3).
Figure 2
Figure 2
Segregation of TYLCV resistant phenotype in the F1S1 population of N. benthamiana LAB and QLD ecotype. The F1S1 individuals from LAB and QLD cross (n=24) was assessed for TYLCV resistance at 3 wpi. In this population, 18 plants (top 3 rows of plants) showed resistant phenotype (R) whereas 6 plants (bottom row) displayed TYLCV susceptible phenotype (S).
Figure 3
Figure 3
Identification of Ty-1/Ty-3 ortholog in N. benthamiana LAB and QLD genomes. (A) Phylogenetic tree of Ty-1/Ty-3 orthologs in number of different plant species including Arabidopsis thaliana (At), tomato (Sl), potato (St), pepper (Ca), tobacco (Nt) and N. benthamiana (Nb), as well as other RDR genes (RDR1, RDR2 and RDR6) of N. benthamiana and A. thaliana. (B) Identification of early stop codon in the coding sequence of RDR3 in LAB isolate. A deletion of a base contributing to an early introduction of a stop codon (TGA; annotated in Orange) was found on the third exon of the LAB RDR3, which is absent in the RDR3 of QLD ecotype. Accession for each gene is provided in Supplementary Table 1 . The Asterisk (*) Denotes confirmed unfunctional/truncated gene in N. benthamiana.
Figure 4
Figure 4
Mutation of RDR3 or RDR5 genes in QLD has no effect on TYLCV resistance. (A) Sequence of the CRISPR-Cas9 induced RDR3 and RDR5 mutant lines (rdr3: Q3-1 and Q3-3, and rdr5: Q5-1 and Q5-6) with edits on the two target sites (blue arrow). (B) The QLD rdr3 and rdr5 plants, showed no disease symptom upon TYLCV inoculation, whereas LAB WT plant displaying the typical TYLCV infection symptoms at 3 wpi.
Figure 5
Figure 5
TMV and TYLCV challenge of the QLD RDR1 knockout mutant. (A) Disease symptoms observed at 3 different timepoints in LAB and QLD plants after TMV inoculation. LAB WT and QLD rdr1 show more pronounced disease symptoms than QLD WT. (B) Systemic accumulation of TYLCV in QLD rdr1 mutant and respective WT plants at 3 wpi (n=6 for the rdr1 mutant (3 plants each from the two independent lines), and n=3 for each wild-type plants.). (C) Accumulation of TYLCV in the apical tissue of rdr1/rdr3 double mutant of QLD and respective WT plants at 3 wpi (n=3, for each line).
Figure 6
Figure 6
Sequence alignment of Pelota proteins in N. benthamiana and tomato. Sequence analysis identified 2 copies of a Pelota ortholog in both LAB and QLD, with one gene in QLD, NbQPelota b, which is missing the first 141 amino acid. The 16th amino acid (blue) is responsible for the resistance in tomato line TY172 (Glycine16), whereas all full-length N. benthamiana Pelota orthologs have Valine16, which is found in the susceptible tomato line, M82.
Figure 7
Figure 7
Susceptibility to TYDV in N. benthamiana LAB, QLD and its F1 progeny. Plants at 3 weeks post Agroinfiltration with TYDV infectious clone (from left: LAB, F1 and QLD plants) and uninoculated control (most right; one plant from each genotype; from top: LAB, F1 and QLD).
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