Host factors involved in potyvirus cell-to-cell movement: new sources of resistance? – PotyMove

Based on a unique consortium with high complementary and transdisciplinary skills (virology, biochemistry, PD proteomics, and genetics), the PotyMove project aims at identifying membrane-associated and/or PD proteins that interact with these three key viral proteins. In parallel, a high throughput genetic approach complementary to PD proteomics will be performed: using forward genetic based on natural diversity or induced diversity, and on quantitative phenotyping, we will identify candidate genes involved in cell-to-cell movement. Furthermore, the PotyMove project aims at providing experimental evidence that the identified candidate genes can afford a broad-range durable resistance to potyviruses and can be transferred to a crop species, tomato, using genome editing strategy. This will be applied to the two initial available candidates, Remorin and PrepP, at the beginning of the PotyMove project.

The role in TuMV movement of the two available candidates at the beginning of the project (PreP et REM) is undertaken through fine quantitative phenotyping of TuMV-GFP infection using a GFP-Cam system (see publication Bastet et al., 2018).
After having demonstrated that each viral MP protein can be used as baits in the SUY2H system, a first screening of an Arabidopsis SUY2H cDNA library allowed the identification of 68 candidates among which 12 were selected for functional validation.
In order to identify other candidate genes potentially involved (directly or indirectly) in potyvirus movement, forward genetic approaches based on natural diversity or induced diversity screening of Arabidopsis were performed. A genomic region of chromosome 1 (spanning around 60 genes, 18 presenting a non-silent mutation between the 2 parents susceptible vs resistant) was identified. In order to narrow this region, F2 genotyping is ongoing and F3 generation is under construction to be phenotyped. The screening of an Arabidopsis KO-eIFiso4E EMS-mutagenized population with a resistance-breaking TuMV isolate, was performed. Using the large-scale inoculation method developed (based on massive agro-inoculation of TuMV-GFP) combined with the GFP-camera imaging, more than 4000 mutagenised Arabidopsis were screened for resistance to a Resistance-Breaking (RB) TuMV-GFP. Ten independent families were collected, showing at least partial resistance to the RB TuMV-GFP. F2 mapping populations are currently being developed for 5 mutations. They will be next phenotyped for resistance in order to design resistance and susceptible bulks, allowing mutant characterization though NGS.

The three viral movement proteins (MP), the negative control (GFP) and the positive control (2B movement protein of GFLV, known to recruit PDLP1, a PD marker) were successfully expressed in E. coli as GST-fusions. PD proteins were obtained by purifying PD channels from A. thaliana cell suspensions. The GST-MP baits will be used to pull-down potential interactors from the PD-enriched fractions.
To study the impact of MP expression or TuMV infection on PD composition of Arabidopsis, transgenic Arabidopsis cell suspensions will be obtained, where MPs or TuMV-GFP expression is inducible. We will then perform a comparative study of the PD-proteome, in two conditions: “healthy” and “infected” (i.e. after induction of MP or TuMV-GFP expression).
For functional validation of the candidates in potyvirus movement, a first quantitative phenotyping will be done, following infection with a TuMV-GFP clone using the GFPCam system. Second, to check whether there are differences in the local spread of the virus between WT and KO plants, we will use a TuMV tool that contains a dual reporter cassette allowing to discriminate between the site of primary infection foci (cells doubly labelled) from secondary infection foci (where cells express only one fluorophore after movement of the virus).
Lastly, we aim at benchmarking the candidates for their potential in plant breeding. For this purpose (i) their resistance spectrum to five species of potyviruses will be evaluated (ii) the mutations affecting the identified candidate genes will be combined with a knock out affecting eIFiso4E, to see whether it reduces the level of resistance breaking by TuMV, finally, (iii) in order to provide experimental evidence that the candidate genes identified in Arabidopsis PotyMove can be transferred to crops, we will characterize loss-of-function mutations affecting the ortholog genes in tomato to see whether they are associated with resistance to potyvirus.

A. Bastet, B. Lederer, N. Giovinazzo, X. Arnoux, S. German-Retana, C. Reinbold, V. Brault, D. Garcia, S. Djennane, S. Gersch, O. Lemaire, C. Robaglia and J.L. Gallois. (2018). Trans-species synthetic gene design allows resistance pyramiding and broad spectrum engineering of virus resistance in plants. Plant Biotechnology Journal. pp. 1–13. doi: 10.1111/pbi.12896.

The development and use of cultivars genetically resistant to plant viruses, notoriously difficult to treat and combat, is a critical factor for sustainable agriculture. Potyvirus is one of the largest genera of plant viruses responsible for serious diseases in important vegetable and fruit crops. To invade plants, those obligatory parasites have developed tactics to reroute host cellular functions for their own benefits.
The completion of the viral cycle results from a complex interplay between virus- and host-encoded factors, also called susceptibility factors. In this scheme, absence or non-adequacy of a single susceptibility factor leads to full or partial resistance to viruses. PotyMove partners were among the first to demonstrate this concept of loss-of-susceptibility resistance genes through the identification of eukaryotic initiation factors as key players in plant-potyvirus interactions, and demonstrated the “transferability” of a potential recessive resistance from the model plant Arabidopsis thaliana to crop plants. Considering the high adaptative potential of plant viruses and resulting resistance breakdown, a more extensive screen of host-virus protein-protein interactors will lead to the identification of new host factors conferring recessive resistance to potyviruses. The ability to combine in the same plant, mutations affecting eukaryotic initiation factors and newly identified genes involved in other steps of the viral cycle, will lead to higher durability of the resistance.
In this context, the aim of PotyMove is to identify new plant factors involved in the virus cell-to-cell movement, a key-step of the viral infection. It is considered to be a major putative obstacle to viral exponential expansion in the plant by generating population bottlenecks and thus, an excellent target for resistance: indeed a defection of plant factors required by the virus for its cell-to-cell movement would cause the virus to be confined or restricted to its primary infection focus, thus delaying or preventing its systemic spreading. Also, by increasing the genetic drift, reduced cell-to-cell virus movement could increase resistance durability.
Plasmodesmata (PD) are symplasmic tunnels between cells that are the gateway for plant virus movement. Pant virus genome encodes a class of proteins called Movement Protein that interact with host proteins to modify the PD for cell-to-cell movement. For the potyvirus genus, this key step is still very little documented. In particular, no dedicated movement protein has been identified, but three viral proteins with other known functions have been reported to participate in potyvirus movement.
Based on a unique consortium with high complementary and transdisciplinary skills (virology, biochemistry, PD proteomics, and genetics), the PotyMove project aims at identifying membrane-associated and/or PD proteins that interact with these three key viral proteins. In parallel, a high throughput genetic approach complementary to PD proteomics will be performed: using forward genetic based on natural diversity or induced diversity, and on quantitative phenotyping, we will identify candidate genes involved in cell-to-cell movement. Furthermore, the PotyMove project aims at providing experimental evidence that the identified candidate genes can afford a broad-range durable resistance to potyviruses and can be transferred to a crop species, tomato, using genome editing strategy. This will be applied to the two initial available candidates, Remorin and PrepP, at the beginning of the PotyMove project.
The potential higher durability of those new resistance genes will rely on the fact that they target viral movement proteins known to be under evolutionary constraint and can be further used in crop species for gene pyramiding with previously identified translation initiation factors, in order to implement multifaceted, durable and consumer-acceptable resistances to virus infection.