Scientists at the Delhi-based National Institute of Plant Genome Research (NIPGR) have used CRISPR-Cas9 gene editing technology to increase phosphate uptake and transport in japonica rice varieties. The resulting rice lines had higher seed and panicle numbers, thereby increasing the yield without compromising seed quality. The studies were carried out in a greenhouse.
Phosphorus is an essential mineral for plant growth and development of plants. In case of limited phosphorus availability, crop productivity drops drastically. Even when phosphate fertilizers are used, only about 15-20% are taken up by plants while the balance gets leached out or lost through runoff.
When the recommended amount of phosphate fertilizer was used, yield increased by 20% in gene edited rice lines. However, when only 10% of the recommended dose of phosphate fertilizer was used, yield in gene-edited rice lines increased by 40% compared with the control, says Dr. Jitender Giri from NIPGR, and the corresponding author of a paper published in Plant Biotechnology Journal.
“The purpose was to just demonstrate that even under extreme conditions of using only 10% of the recommended dose, the gene-edited lines showed increased phosphate uptake resulting in 40% higher yield compared with the control group, where the yield reduced sharply,” says Dr. Giri. “But if phosphate fertilizer supply is reduced by 10% or even 30%, it is very likely that the gene-edited lines will still outperform the control plants.”
Rice absorbs phosphate through its roots and transfers it to the shoots. One class of transporters brings phosphate from the soil into the root, while another inorganic phosphate transporter (OsPHO1;2) transfers phosphate from the root to the shoot. The NIPGR researchers restricted their work to the phosphate transporter that transfers phosphate from the root to the shoot. “When the phosphate transporter OsPHO1;2 starts working more, it will create more demand for phosphate in the root. When this happens, the root-bound transporters will bring more phosphate from soil into the root,” he explains. “We already know there is a negative regulator that controls the expression of the phosphate transporter in the model plant Arabidopsis. But what’s happening in rice was not known till now.”
Identification, removal of the repressor
Through in silico and DNA-protein interaction studies, NIPGR researchers identified the repressor (OsWRKY6) and demonstrated that the repressor physically binds to the promoter. To verify if the repressor was indeed reducing the expression of the phosphate transporter, they silenced the repressor by knocking it out by using the CRISPR-Cas9 gene editing tool. When the repressor was knocked out, the expression of the phosphate transporter (OsPHO1;2) increased significantly.
The increased expression of the transporter should have ideally led to more yield. But instead, the gene-edited rice lines fared poorly compared with the control. “This was unexpected. We figured out that the repressor was also needed for other functions in the plant. While knocking out the repressor gene completely helped in removing the repression of the phosphate transporter thereby increasing the levels of phosphate in the shoot, we were also removing some essential functions regulated by the repressor,” Dr. Giri explains.
Removing the binding site
The researchers then identified the site where the repressor actually binds to the promoter. The binding site in the promoter is a very short sequence of just 30 base pairs. Again CRISPR-Cas9 was used to remove the binding side of the repressor on the promoter. “We removed only the binding site and not the repressor itself. So the repressor is present in the plant and continues to execute other vital plant functions,” Dr. Giri explains.
The phosphate transporter (OsPHO1;2) is also regulated by other regulators. By specifically removing only the site where the repressor binds to the promoter, the researchers ensured that the binding sites of other regulators are intact so they can continue to bind to the promoter and regulate its function. Dr. Giri likens it to undertaking a very precise, minimal invasive surgery in the promoter gene.
There was enhanced expression of the promoter in the roots, along with increased shoot phosphate accumulation and improved plant growth the gene-edited rice plants leading to increased transfer of phosphate from the root to shoot, when the binding site of the repressor gene was removed from the phosphate promoter.
Though only the binding site in the promoter found in the shoot was gene-edited, the researchers found that other transporters present in the root surface brought more phosphorus into the root. “The roots start behaving like a sink by absorbing more phosphate from the soil, and this phosphate is distributed throughout the plant,” he says. The team found that the gene-edited lines were channelising the extra phosphate absorbed by the roots to produce more seeds by increasing the number of panicles — the fruiting body which bears seeds — leading to an increase in yield by 20%. The researchers analysed the seed size, seed dimension, seed length, starch and phosphate content, and found the seed dimension or seed quality to be normal.
Since the roots of gene-edited plants absorb more phosphate than before, will it become even more necessary to continue using the same amount of phosphate fertilizer? That only about 20% of the phosphate fertilizer applied is taken up by plants because Dr. Giri says that phosphate is very reactive. In alkaline soil, phosphate forms complexes with either calcium or magnesium, and if it is acidic, it forms complexes with iron and aluminium. Since phosphate complexes are insoluble in nature, the transporters found in the root cannot absorb them. “In the case of gene-edited rice, the plants will quickly absorb more phosphate before it combines with aluminium, iron, calcium or magnesium and become insoluble,” he explains.
Testing the hypothesis using japonica
For the study, japonica cultivar Nipponbare was used since making gene-edited lines and transgenics is generally easy with japonica. “Japonica variety is easy to work with; it’s not easy to raise transgenics using indica varieties. It will take more time to generate a sufficient number of gene plants when using Indian cultivars,” Dr. Giri says. “So, we test our hypothesis in the japonica variety because it can be done quickly and more reliably, and then replicate it in Indian indica varieties.”
“It’s a very important scientific advancement,” says Dr. P.V. Shivaprasad of the Epigenetics lab in the National Centre for Biological Sciences (NCBS), Bengaluru, who is not part of the study. “Soil in several parts of India has phosphorus deficiency. When the same modifications are performed in indica rice lines, it will be extremely useful. One must also check the efficacy of phosphate absorption, and how much less phosphate fertilizer can be used without compromising yield in indica lines. Exciting times ahead.”
Off-target events
Activists have raised objections to gene-edited technology on the grounds that the IPRs are held by foreign entities. Dr. Giri says that India is negotiating for a license of the CRISPR-Cas9 technology. CRISPR-Cas9 gene editing technology does not always target only the bases/genes of interest. Off-target events do happen, which is another objection raised by activists.
To address the off-target events problem, Dr. Giri says there are software that predict where an intended gene editing might cause unexpected, unwanted, or even adverse alterations to the genome. “We checked for all off-target genes to check if there are any changes. In our case, we tested the top 10 contender off-target sites and did not find any deletion on those sites,” Dr. Giri says. Before the seeds are actually approved and released, and farmers are allowed to cultivate, efforts will be taken to ensure that the deletion is restricted only to the receptor binding site on the promoter with no off-target effect actually seen, he says. “What we do actually is that we produce a large number of lines and then select the best line and check for off-targets,” Dr. Giri says.
“It is very much possible to eliminate off-target events,” says Dr. Shivaprasad. “There are multiple tools available for guide RNA design that almost eliminate the possibility of off-target events. It is also important to check for off-target regions to ensure that off-target events have not happened. It needs expertise.”
According to Dr. Shivaprasad, there are more than three good in silico tools available to check for off-target events. “Southern blot analysis, particularly junction fragment analysis, is carried out to verify the successful integration or modification of DNA sequences within a genome and to confirm if multiple copies or half copies are not present,” he says.
NIPGR researchers have used tissue culture-based transgenic generation. When plants are produced using tissue culture, even before seeds are produced the plants are tested to check if gene editing has been precise without any off-target events. “Only if the gene editing has been precise with no off-target events will we even allow the plants to grow to the seed stage. The rest are discarded. So whatever plant we grow till the seed stage will always carry the correct gene editing. The seeds coming from that plant and from the progeny will carry the phosphate transporter that has been precisely gene-edited to remove the 30 base pairs that form the binding site for the repressor (OsWRKY6),” Dr. Giri says.
Foreign DNA
The third major objection is the presence of foreign DNA. The Cas9 protein used in CRISPR gene editing is derived from Streptococcus pyogenes bacteria. Therefore, Cas9, which acts as a DNA-cutting enzyme, carries foreign DNA. Foreign DNA also comes from soil bacterium that is used as a vector for delivering the CRISPR-Cas9 components into plant cells.
Dr. Giri claims that the DNA from bacteria is removed in the second generation through a simple Mendelian segregation method, as the plants are tested before growing to the seed stage to know if the gene editing has been precise without any off-target events. “If you have one trait, the next generation will segregate into 3:1, where three will have the foreign DNA, and one will not. In the next generation, foreign DNA free plants are identified and propagated,” he says.
“It is possible to remove the DNA of Agrobacterium tumefaciens — the soil bacterium that is used as a vector for delivering the CRISPR-Cas9 components into plant cells — through the Mendelian segregation method,” confirms Dr. Shivaprasad. When the soil bacterium vector is removed, the S. pyogenes bacterium also gets removed automatically.
India is almost entirely dependent on imports — nearly 4.5 million tonnes of Diammonium phosphate (DAP) — to meet the demand for phosphate fertilizers. The gene-edited technology, if successfully replicated in Indian rice varieties, can possibly contribute towards sustainable agriculture.