Plant biotechnology: Engineering disease resistant plants
Plant pathogens have been a serious challenge to crop production worldwide. External use of chemicals to fight crop diseases is now considered to be detrimental to human health and the environment. Encouragingly, genetic engineering now provides a more sustainable and desirable alternative to boost plant disease resistance.
Do plants have their own immune system?
One of the ways plant immune system acts is by the production of a small phenolic compound salicylic acid (SA). Plant’s pattern-triggered immunity (PTI) is based on the recognition of “microbe-associated molecular patterns (MAMPs) which stimulates SA production.
SA-activated immunity has been extensively studied in the model plant Arabidopsis. Here, a protein called “non-expressor of pathogenesis-related genes 1 (NPR1) serves as a master regulator of such immune response.
Expression of the NPR1 gene is constitutive. Normally, NPR1 protein molecules reside in the cell cytoplasm as oligomers. In response to SA-induced redox changes the NPR1 protein is liberated from a quiescent oligomeric state. Subsequently, the monomers translocate into the cell nucleus.
In the nucleus, NPR1 serves as a coactivator in complex with TGACG-binding (TGA) transcription factors. TGA contains a leucine-zipper motif. The motif is a single a-helix comprising a DNA-binding region and a dimerization region. The NPR1-TGA complex bind to the pathogenesis-related-1 (PR1) gene promoter at the activating sequence-1 (as-1) element. Subsequently, Mediator proteins facilitates NPR1-TGA interaction with RNA polymerase II (RNA pol II) to initiate transcription.
(In eukaryotes, most often regulatory molecules interact with RNA pol II through an “intermediate”. This intermediate may not be a single protein, but a multiprotein complex called Mediator.)
Boosting plant disease resistance
Most often, plant’s own immune system is not sufficiently robust to counter pathogenic onslaught. So, it is imperative to empower the plants with enhanced and durable resistance against a broad range of pathogens. Transformation with genes encoding antimicrobial peptides (AMPs) has been found to be an effective approach to boost plant disease resistance.
AMPs belong to the innate immune system of a multitude of animals and plants. Most of them act by lysing the membrane of invading microorganisms. Two of the well-known AMPs are cecropin and melittin. They are small peptides containing 39 and 26 amino acid residues respectively. Cecropin A was originally isolated from the hemolymph of the giant silk moth, Hyalophora cecropia. Melittin was isolated from bee venom.
When expressed in plants, any of these AMPs has been found to increase host resistance against bacterial infection. Interestingly, a cecropin A-melittin hybrid peptide (CEMA) turned out to be the most effective.
Needless to say, the CEMA gene must be fused to a suitable promoter for RNA polymerase’s transcriptional activity. Constitutive expression of the gene can confer disease resistance on transgenic plants. Nevertheless, high-level of expression of the target gene may have an adverse effect on the growth and yield of the crop. An inducible promoter, such as that regulated by SA-NPR1-TGA, is, therefore, highly preferable. Further, a polyadenylation sequence in the construct ensures that the mRNA is translated into a peptide in plant (eukaryotic) cells.
How are genes transferred to plant cells?
The most widely used strategy for gene transfer to plant cells relies on the soil phytopathogen, Agrobacterium tumefaciens. The bacterium infects wound sites in a variety of plants and causes tumorigenic transformation known as crown gall disease.
Plant tumorigenesis is caused by only virulent Agrobacterium strains. Such strains harbor large (140—235 kbp) tumor-inducing plasmids (Ti-plasmids). A short segment of the Ti-plasmid, called T DNA (transferred DNA) carries the oncogenes required for tumorigenesis. The T-DNA is transferred from the infecting bacterium to the host plant cell. Here, it integrates at an apparently random site of the host genome.
Notably, genes that the T-DNA carries are non-essential for the transfer mechanism and can be replaced with foreign DNA. Transfer-related genes are located in a separate segment of the Ti-plasmid called the vir (virulence) region. Nonetheless, the T-DNA is flanked by 25 bp imperfect direct repeats known as border sequences. While deletion of the right border (R-border) abolishes T-DNA transfer, the left border (L-border) appears to be nonessential.
Therefore, it is evident that the Ti-plasmid is a natural vector for plant genetic engineering. However, to regenerate plants efficiently, its T-DNA needs to be “disarmed” by deleting all the oncogenes. Such a disarmed T-DNA, carrying the CEMA gene expression construct, can remain stably integrated in the plant genome and express the hybrid AMP. As reported, up to four copies of the transgene are integrated at different sites of the host genome. Evidently, a high level of CEMA production in the transgenic plant cells can be expected.
Can plant disease resistance be further boosted?
Additional resistance against even a broader range of pathogens can be conferred on plants by the inclusion of a jasmonic acid (JA)-responsive promoter in the transgene construct. JA is another hormone synthesized in pathogen-invaded plants.
Promoters that respond to JA contain G-box (CACGTG) and GCC-box (AGCCGCC)-box motifs. In particular, the G-box can recruit the transcription factor MYC2. JA-signaling enables MYC2 to interact with RNA pol II through Mediator Med25 to initiate transcription of JA-responsive genes.
As expected, a dual-promoter containing SA- and JA-responsive elements suitably located is able to recruit both TGA and MYC. The use of such a promoter to drive the expression of a hybrid AMP can further boost plant disease resistance. This transgenic strategy has worked well also in tobacco, tomato and potato.