Recombinant Construct for Concurrent DNA and RNA Virus Resistance
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Solution Overview
Problem
Current methods for providing plants with resistance against both DNA and RNA viruses often result in short delays or reduced disease severity, and may introduce marker genes that express foreign proteins, posing ecological and food safety risks.
Innovation Solution
A recombinant construct comprising an intron and exon region is used, where the intron region includes a fragment of an intergenic sequence from a DNA virus promoter and an antisense sequence, linked with a spacer, and the exon region contains a nucleocapsid protein gene of an RNA virus with stop codons, preventing protein expression, and is integrated into a binary vector without a selectable marker gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If marker genes are used to select transgenic plants, then transgenic plants can be identified and selected, but foreign proteins may be expressed causing ecological and food safety risks
Solution Approach 1:
The invention extracts and removes the harmful marker gene component from the transgenic plant system. By using a virus-resistant plant that naturally lacks foreign marker genes, the solution eliminates the source of harmful protein expression while preserving the ability to identify and select transgenic plants through viral resistance phenotyping
Solution Approach 2:
The invention converts the plant's natural viral susceptibility into a beneficial selection mechanism. Plants are engineered to express viral resistance, and those that successfully integrate the resistance gene can be selected by their ability to resist viral infection, thereby converting what would normally be a harmful viral interaction into a useful selection tool without requiring foreign marker genes
2Reliability
If PTGS approach is used for DNA virus control, then some resistance is achieved, but only short delay in symptom development or reduced disease severity results
Solution Approach 1:
The invention applies preliminary action by implementing RNA-directed DNA methylation (RdDM) before viral infection occurs. This epigenetic modification pre-marks the viral DNA sequences for silencing, establishing a preventive defense mechanism that activates upon viral infection rather than relying on post-infection PTGS alone, thereby achieving more effective and timely resistance
3Reliability
If foreign genes are introduced into plants, then virus resistance is provided, but the foreign genes may be expressed as foreign proteins in plant
Solution Approach 1:
The invention extracts and removes the problematic protein-coding sequences from the introduced genetic material. By using viral intergenic region sequences that contain promoter elements and targeting sequences but lack complete open reading frames, the solution enables viral DNA recognition and methylation while preventing the expression of harmful foreign proteins
Solution Approach 2:
The invention uses viral intergenic region sequences as intermediary elements that mediate between the plant's defense system and the viral genome. These sequences serve as recognition targets for RNA-directed DNA methylation without being translated into proteins, acting as a safe interface that enables resistance without foreign protein expression
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
This approach induces RNA-directed DNA methylation and post-transcriptional gene silencing, providing effective resistance against both DNA and RNA viruses without expressing foreign proteins, thereby reducing ecological and food safety concerns.
Implementation Method 1
TGS occurs in nuclei via RNA-directed DNA methylation (RdDM) at CG, CHG and CHH sequence contexts
Implementation Method 2
PTGS operates in the cytoplasm through mRNA cleavage or inhibition of translation
Data Source
AI summary
Provided is a strategy for generating transgenic plants with concurrent resistance to DNA and RNA viruses at one construction, so as to develop an RNA-directed DNA methylation (RdDM) transgenic system using a hairpin construct of Ageratum yellow vein virus (AYVV) promoter region residing in an intron to resist DNA virus infection by RdDM. Furthermore, the hairpin construct of the AYVV promoter region coupled with an untranslatable nucleocapsid protein (NP) fragment of Melon yellow sport virus (MYSV) is created to induce post-transcriptional gene silencing (PTGS) against MYSV. A method for providing transgenic plants conferring concurrent resistance to both AYVV and MYSV for control of DNA and RNA virus at the same time, and underlying RdDM and PTGS mechanisms, respectively, is also provided.