Genetically Modified Golden Bean Mosaic Virus Resistant BeansMichael Xie12/2/14

What's the big deal about beans?

• Common Bean (Phaseolus vulgaris L.) is a popular food that is a good source of proteins, carbohydrates, and vitamins in many areas of the world, especially in developing countries.

• Latin America is the largest producer (5.5 million tons), with Africa in second (2.5million tons).

• Estimated 90,000-280,000 tons of beans are lost each year to Bean Golden Mosaic Virus (BGMV) in Latin America.

• This drove the Brazilian company, Embrapa to engineer a genetically resistant commercial bean crop.

Bean Golden Mosaic Virus (BGMV)• The virus is transmitted by the whitefly (Bemisia tabaci)• It infects the common bean and causes them to have a yellow-green

mosaic pattern of leaves, stunted growth, and distorted pods

BGMV Structure• Composed of 2 ssDNA molecules• DNA-A DNA-B AC1-Replication Transcription Factor (REP)BC1-Movement Protein (MP) AC3-Replication Enhancer Protein BV1-Nuclear Shuttle Protein AC2-Transcription Regulatory Protein (TrAP) AV1-Paticle encapsidation

RNAi-mediated resistance• Target Gene to be silenced: The

AC1 ( Viral Replication Gene)• Once the transgenic bean line has

been created:• The transgene encoding

complementary RNA will be expressed and bind to AC1 to form a dsRNA structure

• dnRNA will cleaved by Dicer (Rnase III) into small interfering RNA

• Those siRNA will bind to target mRNA (AC1) and be targeted for degraded by the RISC protein complex

Engineering the TransgeneGeneration of AC1 fragments from using AC1 primers, Viral DNA, and PCRFragments are inserted into a pGEM-T easy vector to clone the PCR products The fragments are placed into the pAC321 vector that has the imazpyr resistance gene (ahas) selectable markerPdk is the Flaveria trinervia intron and is used as the probe marker during southern blotting

Bean Transformation• Mature seeds undergo micro-projectile bombardment of their

embryonic axes with the transgene vector• This was done by a high-pressure, helium driven particle

acceleration device

Seeds are then grown in a culture containing imazapyr

Screening for the Transgene Transgene copies did not independently segregateEvidence in XhoI digested Southern Blot Analysis

Screening for RNAi • Seedlings were infected by 200/300 whiteflies carrying the

virus for a 6 day period and a 12 day period• Quantitative PCR reveals Viral DNA at 6, and none at 12• Northern blot analysis:

Results

Concerns• Has been Approved by the National Biosafety Technical

Commission for human consumption and field production

• Unfair for Small Scale Farmers?• GMO’s are unhealthy to eat?• Reduction of Bio-Diversity?• Increased Virus Resistance?