BPMV Vectors for Soybean Gene Expression and Silencing

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Solution Overview

Problem

Current plant viral vectors, particularly for soybean, face limitations in systemic expression of foreign proteins and gene silencing, with previous Bean pod mottle virus (BPMV) vectors requiring foreign sequences to be in-frame with the viral polyprotein, restricting design flexibility and only allowing single gene expression.

Innovation Solution

Development of novel BPMV vectors with insertion sites after the RNA2 translation stop codon, enabling non-coding sequence insertion and simultaneous expression of foreign genes, using Foot and Mouth Disease Virus 2A proteinase for efficient protein excision, and modifying RNA1 for moderate symptom phenotypes to improve vector performance.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If foreign sequences are inserted in-frame with the viral polyprotein in previous BPMV vectors, then the viral polyprotein can be properly synthesized, but the design flexibility is restricted and only single gene expression is allowed

Engineering Contradiction:

Improvedesign flexibility and gene expression capacityVSAvoidvector structure complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The vector design separates the foreign gene insertion site from the viral polyprotein coding region. The foreign sequence is inserted after the RNA2 translation stop codon rather than within the polyprotein ORF, allowing independent expression without disrupting viral polyprotein synthesis. This segmentation enables multiple genes to be expressed simultaneously while maintaining viral functionality.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A protease cleavage site (Foot and Mouth Disease Virus 2A proteinase) is introduced as an intermediary element between the viral coat protein gene and the foreign gene. This cleavage site acts as a mediator that allows efficient separation of the foreign protein from the viral polyprotein, enabling flexible gene expression without compromising the integrity of the viral replication system.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If traditional VIGS vectors are used, then gene silencing can be achieved, but the requirement for in-frame reading restricts the types of sequences that can be targeted

Engineering Contradiction:

Improvesequence targeting capabilityVSAvoidvector design simplicity

Core Design Contradiction:

Adaptability or versatilityVSEase of operation

Solution Approach 1:

Instead of requiring the target sequence to be in-frame with the viral polyprotein (traditional approach), the invention inverts the approach by placing the foreign sequence after the stop codon. This allows non-coding sequences, promoter regions, and any genomic DNA fragment to be targeted for VIGS without translation requirements, greatly expanding sequence targeting capability.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The invention changes the fundamental parameter of sequence expression from translational (in-frame coding) to transcriptional (RNA-mediated silencing). By placing insertion sites after the stop codon, the system allows any sequence to be transcribed into RNA that can trigger silencing, without requiring the sequence to code for a functional protein.

Inventive Principle:

Principle #35Parameter changes

3Reliability

If RNA1 is modified for moderate symptom phenotypes, then vector performance and plant survival are improved, but the viral pathogenicity is reduced

Engineering Contradiction:

Improvevector performance and plant survivalVSAvoidviral symptom severity

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

Specific amino acid substitutions are introduced in the RNA1-encoded proteins (helicase and coat protein) to modulate viral pathogenicity. The changes in molecular structure of these proteins result in altered viral-host interactions, producing moderate symptoms that balance vector performance with plant survival and productivity.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The new BPMV vectors facilitate high-throughput applications, allow for the expression of multiple genes, and achieve efficient RNA silencing without the need for in-frame reading, enhancing the utility for soybean functional genomics and protein production.

Implementation Method 1

using Foot and Mouth Disease Virus 2A proteinase for efficient protein excision

Methodology Applied

Scientific EffectProteolytic cleavage: Enzyme

Implementation Method 2

achieve efficient RNA silencing without the need for in-frame reading, enhancing the utility for soybean functional genomics

Methodology Applied

Scientific EffectRNA interference:

Data Source

PatentUS8569579B2BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes

Publication Date: 2013.10.29 IOWA STATE UNIV RES FOUND INC

AI summary

The invention provides Bean pod mottle virus (BPMV) vectors useful for expression of heterologous proteins in plants such as soybean. The BPMV vectors are also useful for virus-induced gene silencing. The vectors of the invention include modifications of BPMV RNA1 sequences so that infection with the vectors produces only moderate symptoms. The vectors also comprise novel RNA2 vectors which specifically provide for non-translated VIGS constructs and further which do not require in frame insertion of heterologous sequences to be expressed.