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Method of increasing resistance against soybean rust in transgenic plants by increasing the scopoletin content

a technology of scopoletin and soybean rust, which is applied in the direction of plant peptides, biochemistry apparatus and processes, enzymes, etc., can solve the problems of host serious hampered in development or die-off, high susceptibility to epidemic-like spreading of diseases, and high yield reduction, so as to increase the resistance against fungal pathogens, increase the content of scopoletin, and increase the expression of f6h1

Active Publication Date: 2018-01-11
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about finding a way to make plants resistant to fungal infections, particularly soybean rust, Fusarium graminearum, and Fusarium verticillioides. The method involves increasing the amount of a compound called scopoletin in plants. This can be achieved by either increasing the natural expression of a specific protein called F6H1 or by using a formulation or solution containing scopoletin. The invention also includes recombinant expression vector constructs that contain a sequence that is similar to F6H1 protein.

Problems solved by technology

Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases.
The result is markedly reduced yields.
The pathogen survives, and may build up reproduction structures, while the host is seriously hampered in development or dies off.
They cause seed rots, seedling blights as well as root rots, stalk rots and ear rots.
After a very short establishment phase the Fusarium fungi start to secrete mycotoxins such as trichothecenes, zearalenone and fusaric acid into the infected host tissues leading to cell death and maceration of the infected tissue.
Nourishing from dead tissue the fungus then starts to spread through the infected plant leading to severe yield losses and decreases in quality of the harvested grain.
Interestingly, scopoletin biosynthesis seems to be lost in several economically important crops (e.g. Glycine max, Zea mays, Triticum aestivum, Oryza sativa etc.), indicating that the ability to synthesize this antimicrobial substance might have been lost during breeding.
Soy plants with resistance to the entire spectrum of the isolates are not available.
Therefore only little progress was made in increasing resistance against Fusarium by breeding.
However, the problem to cope with soybean rust which infects the mesophyll or Fusarium fungi that infect inaccessible inner tissues remains unsolved.

Method used

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  • Method of increasing resistance against soybean rust in transgenic plants by increasing the scopoletin content
  • Method of increasing resistance against soybean rust in transgenic plants by increasing the scopoletin content
  • Method of increasing resistance against soybean rust in transgenic plants by increasing the scopoletin content

Examples

Experimental program
Comparison scheme
Effect test

example 1

ethods

[0323]The chemical synthesis of oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977).

Example 2: Cloning of Overexpression Vector Constructs for Transient N. benthamiana Transformation

[032...

example 4

ng Abundance of Gene Transcripts

[0335]Total RNA was extracted from leaves of the described Arabidopsis mutants as described by Chomczynski and Sacchi (1987). 1 μg RNA was transcribed to cDNA using random primers (9-mers) and RevertAid™ reverse transcriptase (Fermentas) according to manufacturer's instructions. Accumulation of gene transcripts was quantified in an ABI7300 using SYBR green (Invitrogen) at the following conditions for RT-qPCR: 50° C. for 2 min, 95° C. for 10 min, 95° C. for 15 s, 60° C. for 1 min, 95° C. for 15 s, 60° C. for 1 min, and 95° C. for 15 s (the third and fourth steps were repeated 40 times).

Primers specifically hybridizing to F6H1 gene (SEQ ID No 1):F6H1_RT_F:(SEQ ID NO: 81)5′-CTCAGCCTCTTCTTTGTCTC-3F6H1_RT_R:(SEQ ID NO: 82)5′-AAGCCTCCTCACCATCTTC-3′Primers specifically hybridizing to CCoAOMT1 (SEQ ID No 3):CCoAOMT1_RT_F:(SEQ ID NO: 83)5′-ATGGCGACGACAACAACAGAAGC-3CCoAOMT1_RT_R:(SEQ ID NO: 84)5′-GCCAATCACTCCTCCAATTTTCACA-3′Primers specifically hybridizing to A...

example 5

Germination Tests

Example 5a Growth Inhibition of Phakopsora pachyrhizi

[0337]Spores of Phakopsora pachyrhizi were resuspended in H2O supplemented with Tween-20 and 10 μM, 100 μM, 500 μM and 1 mM scopoletin. Spores of Phakopsora pachyrhizi resuspended in H2O supplemented with Tween-20 were used as control. All resuspended spores were transferred onto glass slides. After six hours incubation time the ASR spores were germinated and started to form appressoria.

[0338]The germination rate and appressoria formation rate was determined by quantitative microscopic analysis. Spores showing a visible germtube formation but no thickening of the germ tube tip were counted as “germinated”, whereas the presence of a thickened germ tube tip indicated the formation of an appressoria (FIG. 14a).

[0339]Application of scopoletin to ASR spores decreases the germination and appressoria formation in a dose dependent manner. At 1 mM concentration scopoletin completely abolishes spore germination in-vitro.

Ex...

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Abstract

A method for increasing fungal resistance in a plant, a plant part, or a plant cell wherein the method comprises the step of increasing the production and / or accumulation of scopoletin and / or a derivative thereof in the plant, plant part, or plant cell in comparison to a wild type plant, wild type plant part, or wild type plant cell.

Description

SUMMARY OF THE INVENTION[0001]The present invention relates to a method of increasing resistance against fungal pathogens, in particular soybean rust and / or Fusarium graminearum and / or Fusarium verticillioides, in plants, plant parts, and / or plant cells. This is achieved by increasing the content of scopoletin and / or a derivative thereof, in particular by increasing the expression of F6H1 in a plant, plant part and / or plant cell. This can also be achieved by application of a formulation or solution containing scopoletin and / or a derivative thereof.[0002]Furthermore, the invention relates to recombinant expression vector constructs comprising a sequence that is identical or homologous to a sequence encoding F6H1 protein.BACKGROUND OF THE INVENTION[0003]The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of disease...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82C12N9/02C07K14/415C12N9/10
CPCC12N15/8282C07K14/415C12N9/0071C12N9/1007C12N9/1048C12Y114/11C12Y201/01104
Inventor SCHULTHEISS, HOLGERCONRATH, UWELANGENBACH, CASPAR
Owner BASF PLANT SCI GMBH
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