DURABLE RHIZOMANIA RESISTANCE

Abstract

A method to predict and modulate the Rz2 resistance towards Beet necrotic yellowing vein virus (BNYVV) is based on the identification of variance in TGB1 from BNYVV, isolated variant TGB1 and corresponding uses, as well as the use of Rz2 sugar beets for soils infected with the beet soil-borne mosaic virus.

Claims

1. A method to modulate the resistance towards Beet necrotic yellowing vein virus (BNYVV) comprising: obtaining a plant or a plant tissue expressing a functional Rz2 protein against BNYVV, obtaining a variant BNYVV TGB1 protein having between 90% and 99% of identity with SEQ. ID NO:2 and/or with SEQ. ID NO:3 and/or with SEQ. ID NO:4, measuring interaction between the said functional Rz2 protein (SEQ. ID NO:1) and the said variant TGB1 protein, obtaining a plant or a plant tissue expressing a variant Rz2 protein, measuring interaction between the said variant Rz2 protein and the said variant TGB1 protein and selecting a variant Rz2 protein displaying an increased interaction with said variant TGB1 protein by comparison to the interaction of said functional Rz2 protein (SEQ. ID NO:1) with said variant TGB1 protein.

2. A method to monitor resistance of a sugar beet plant towards Beet necrotic yellowing vein virus (BNYVV), said sugar beet plant expressing a functional Rz2 protein, comprising isolating a TGB1 (SEQ. ID NO:2) protein variant from said BNYVV and measuring interaction between said functional Rz2 protein and said TGB1 protein variant of said BNYVV.

3. The method of claim 1, wherein the interaction between Rz2 protein or a variant thereof and TGB1 protein or of a variant thereof is measured at the protein level, by pull-down, by yeast two hybrid, by fluorescence complementation or by FRET, or in vivo, by measuring a resistance response upon addition of said TGB1 protein or of said variant of TGB1 protein to a plant or a plant part expressing said functional Rz2 or said variant Rz2.

4. The method according to claim 1, wherein the functional Rz2 protein or the variant of said functional Rz2 protein shares at least 90% of identity with SEQ. ID NO:1 over the full-length of said sequence.

5. The method according to claim 1, wherein the variant TGB1 protein is obtained from BNYVV isolated from soils with long-term cultivation of sugar beet expressing a functional Rz2 protein.

6. A method of using a TGB1 protein (SEQ. ID NO:2) or of a variant thereof from Beet necrotic yellowing vein virus (BNYVV) comprising monitoring occurrence of Rz2 resistance-breaking mutants, said TGB1 protein variant having between 90% and 99% of identity with SEQ. ID NO:2 and/or with SEQ. ID NO:3 and/or with SEQ. ID NO:4, said identity being measured over at least 100 consecutive amino acids.

7. The method of claim 6, wherein the variant TGB1 is isolated from soils with long-term cultivation of sugar beets expressing a functional Rz2 protein.

8. An isolated TGB1 obtained from Beet necrotic yellowing vein virus (BNYVV) from soils with long-term cultivation of sugar beets expressing a functional Rz2 protein.

9. A method of using the isolated TGB1 of claim 8 comprising monitoring the Rz2 resistance towards BNYVV.

10. A method of using a sugar beet plant or seed expressing a functional Rz2 protein for soils infected with a beet soil-borne mosaic virus or for soils infected with the beet soil-borne virus.

11. The method of claim 10, wherein the functional Rz2 protein shares at least 90% of identity with SEQ. ID NO:1 over the full-length of said sequence.

12. A method of using a sugar beet plant or seed expressing a functional Rz2 protein (SEQ. ID NO:1) for soils infected with a beet soil-borne mosaic virus or for soils infected with the beet soil-borne virus.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] FIG. 1: Quantification of viral content in sugar beet plants after vortex-inoculation of BNYVV and of BSBMV.

[0058] FIG. 2: Identification of the avirulence gene for Rz2 by transient expression.

EXAMPLES

Example 1

[0059] BSBMV Infection is Controlled by Rz2, but not by Rz1.

[0060] Generation of the Viruses

[0061] Viruses have been obtained from infected soils and propagated as well-known in the art. For instance, B. macrocarpa have been infected with either BNYVV or BSBMV. Then, the sap from systemically infected leaves was produced and used to infect young sugar beet plants, for instance by mechanical inoculation.

[0062] Infection of Sugar Beet Plants

[0063] Existing varieties of sugar beet plants with either Rz1, Rz2 (SES VanderHave, Belgium) or no resistance towards rhizomania (susc; two different varieties) have been inoculated with either BNYVV (an isolate with 4 RNAs) or BSBMV by vortex inoculation.

[0064] The abundance of the virus in the plant (infected or not) is estimated by ELISA quantification of the viral coat protein.

[0065] Rz1 and Rz2 efficiently control the infection with BNYVV (FIG. 1). Rz2, in addition, provides a strong protection against infection with BSBMV or BSBV.

[0066] Moreover, the clones that have been developed are shown to correctly reflect the underlying physiology, and can thus be used for further experiments, such as infection experiments of Rz2-containing plants.

Example 2

[0067] Identification of the Avirulence Protein of BNYVV and of BSBMV

[0068] The inventors have expressed several constructs of BNYVV and/or of BSBMV in Beta plants expressing a functional Rz2 protein and no functional Rz1 and checked in plantar, after 3,3-diaminobenzidine (DAB) staining, for an immune response.

[0069] DAB Staining

[0070] Rationale: H.sub.2O.sub.2 is produced during resistance response, such as a hypersensitive (immune) reaction. The polymerization product of DAB in contact with H.sub.2O.sub.2 results in a strong-reddish brown color, which is visible to the naked eye.

[0071] Protocol: 0,1% DAB-PBS buffer is infiltrated by vacuum into detached leaf-samples. Incubation for at least 3 hrs; boiling of samples in 96% ethanol to bleach samples.

[0072] The expression of RNAs 1-4 of BNYVV and of BSBMV causes a major resistance response in Rz2 (not Rz1) plants and no resistance response in non-Rz2 plants (FIG. 2, left column).

[0073] A similar resistance response was observed in Rz2 plants infiltrated with only RNA 1-2 of BNYVV.

[0074] However, the expression of RNA 1 alone did not elicit immune resistance response in Rz2 plants.

[0075] Then the inventors identified that only TGB1 (SEQ. ID.NO:2) of BNYVV (and not the other proteins expressed by this virus) caused immune resistance response in Rz2 plants (FIG. 2, right column). This protein is necessary and sufficient for this phenomenon in these plants.