Genetic Underpinnings of Heat Resistant Potatoes

Identifying the genetic basis of a particular trait is the building block on which molecular and transgenic breeding strategies build upon. How we connect the dots between a genotype and trait is the basis of a past post “How do we find genes related to traits? A review of bulked sample analysis”.

Despite only being behind rice and wheat as the most important crop worldwide, quantitative trait loci (QTLs) for heat tolerance in potatoes have not, until recently, been identified.

Potatoes are particularly sensitive to heat, with temperatures above the ideal maximum of 22°C resulting in significant decreases in growth and yield. The higher temperatures have a number of effects:

1. Strongly suppresses tuberisation;
2. Reduces the proportion of carbon assimilated for tuber starch;
3. Reduces chlorophyll levels and CO2 fixation rates;
4. Halts tuber dormancy resulting in early sprouting and secondary growths.

A recent study in the Plant Biotechnology Journal sought to identify QTLs for heat tolerance by comparing differences in gene expression and tuber yield between progeny of a biparental diploid potato (06H1) when grown at 22°C and 28°C.

Results

Temperature response analysis and gene identification

Single node cuttings were used as a method to quickly induce tuberisation of subject potato plants, allowing a faster assessment of tuber yield (see this article discussing its use – paywalled unfortunately). 170 genotypes from the 06H1 population were analysed using 6 nodal cuttings from each to assess tuber yield under 22°C and another 6 for each genotype assessed at 28°C. On average, tuber yield at the lower temperature (1.017 grams) was greater than for nodes grown at the higher temperature (0.285 grams). The difference in performance was used in combination with single nucleotide polymorphism (SNP) maps of the 06H1 genome to identify genetic markers which may be connected to the genes which alter expression rates at the two temperatures.

Figure 1 from article. Comparison of tuber fresh weight yield when grown at 22°C and 28°C.

Statistical analysis of the differences in expression levels at each marker found 3 markers which may be connected to the difference in phenotype. Using the Potato Genome Browser the researchers browsed the list of genes connected to the markers for suitable gene candidates for further research. Of the three markers, one marker contained a possible gene candidate, that encoding Heat Shock Cognate 70, a gene that has been suggested as involved in the heat tolerance in cabbage and potato.

With a candidate gene identified, the gene was isolated at amplified for analysis of allelic differences within the gene, of which 4 were identified and named A1, A2, A3 and A4. Nodes of potatoes containing each the four alleles were assessed for tuber yield, with allele A2 having the greatest fresh weight at both 22°C and 28°C and combined A2A3 genotypes having the highest yield. Gene transcript levels were assessed in plants grown at both temperatures which showed that the HSc70 gene was upregulated in A2A3 plants but not in the A1A4 plants, while the HSc70 allele up-regulated in plants grown at the warmer temperature were all from the A2 allele.

Analysis of the amino acids of each of the alleles themselves found that the A2 and A3 proteins contained an additional C-terminal sequence of KIEEVD (thought to be involved in interactions with co-chaperones), while no other variations were unique in a single allele.

What was unique to the A2 allele was a difference in the promoter sequence where a run of ten TA repeats was found as opposed to a run of four TA repeats in the promoters of the other repeats. The researchers later hypothesize that the greater number of TA repeats may result in enhanced expression levels, a hypothesis based on previous research in yeast, although the specific effect of the repeat TA sequence is not known.

Confirming the effects of the HSc70 A2 allele

To test the A2 allele and its promoter sequence, the gene and upstream component was transferred into Nicotiana benthamiana plants and subjected to 45°C heat in a growth chamber. After 24 hours the expression of the HSc70 gene was 7 times greater than in the control plants and cell membrane injury in the transgenic plants were significantly lower.

The unique promoter was then targeted for further analysis. The A2 allele was put downstream of a series of promoters with TA repeats varying between four and ten times. After being exposed to the higher temperatures the level of HSc70 transcription was measured. Alleles with eight to ten TA repeats in the promoter showed statistically significant elevated transcript levels compared to alleles with four to six TA repeats in the promoter.

Figure 6 from article. a) HSc70 expression levels for promoters containing 4, 6, 8 and 10 TA repeats and b) cell membrane injury percentage for plants containing the same numbers of TA repeats.

A2 allele-containing plants with significantly increased expression under 28°C heat were selected for analysis under 40°C conditions for 24 hours in comparison to wild-type plants. Extracts of plants after 4 hours at this temperature showed HSc70 expression being enhanced by up to 50 times more than in control plants. However, after 24 hours the transcript levels fell back to that seen prior to being subjected to the extreme heat. The A2 allele-containing lines has significantly less cell membrane damage compared to the wild-type.

Finally, the ability of the A2 allele to protect tuber yield at elevated temperatures was tested. Using the same nodal cutting tuber yield assay as before, A2 allele containing plants were compared with wild Desiree potato plants when grown at 20°C showed little difference in tuber yield. However, at 28°C the Desiree plants reduced yield by 75% compared to yield at 22°C. By comparison, transgenic lines had significantly lower yield reductions, with some lines have twice the fresh and dry weight yield as the wild type.

Conclusion

The identification of the heat shock cognate protein 70 within the potato genome and the quantification of the effects of the specific A2 allele demonstrates the significance of quality genome research, genome annotation and the ability of powerful molecular tools to seek out expression variances that allow us to pinpoint the basis of variances in phenotype. Coupled with well prepared and executed experiments, the development of these tools will continue to enable us to extract information that will assist us to assist crops and food production.

Whether it is through transgenic technology or molecular breeding, the identification of genes such as the one discovered in this study will assist food production meet the challenges of a changing climate.

Identifying the specific allele of the HSc70 protein that contributes to its resistance to the negative effects of elevated temperature is surely a big step into future-proofing the security of this important crop.

Activation and Repression of Transcription Factors via a Synthetic Construct in Arabidopsis thaliana

Being able to turn genes on and off as required is a significant goal in genetic manipulation. Synthetically constructed gene circuits which turn on and off in response to specific activators have previously been demonstrated and the ability knock out specific genes with such technology as CRISPR/Cas 9 has made gene manipulation easier and cheaper.

But the purpose of this particular article in the Plant Biolotechnology Journal (open-access = awesome) was focused on manipulating the mechanics of gene transcription repression to turn off gene repressors (and activate expression) and then reverse the process in subsequent generations as required.

Gene Transcription

Transcription factors, the proteins that form part of the machinery that transcribes DNA sequences into RNA, contain motifs that allow for them to be activated and deactivated. Regulation of gene transcription using the activation and deactivation of transcription factors is a necessity in all cells, allowing the expression of genes to be limited to particular moments within a cycle or at different stages of development.

The examples used in this study when demonstrating the novel bit of tech the researchers developed to force the repression and activation of different genes illustrate the need for this function well. One gene, MYB80, codes a transcription factor necessary for pollen development and the programmed cell death (PCD) of the tapetal in plant anthers. If allowed to transcribe one of its targeted genes unregulated, PCD occurs too early and the male flowers are rendered sterile, meaning the transcription must be regulated if the plant is to develop properly. This consequence was discovered in an earlier study when a mutant of the transcription factor was created which deleted part of the encoding gene which resulted in the loss of the regulatory motifs, likely meaning the the transcription factor was unhindered in transcribing its target genes.

Another transcription factor studied by the researchers was that encoded by WUSCHEL (WUS), required for the development of the organising centre beneath the stem cells in the meristem. Regulation of WUS activity is required to create distinct cell boundaries and a lack of regulation leads to disorganised growth and male sterility. Similar to MYB80, the transcription factor has a number of regulatory motifs at the C-terminal end of the protein which interact with corepressors.

Manipulating the function of transcription factors to regulate gene transcription is therefore another potential method of turning genes on and off to test the knock-on effects and potentially alter and improve plant characteristics.

Synthetically Regulating the Transcription Factors

How these regulatory motifs inhibit gene transcription, and how we might be able to cause the same inhibition, was the focus of this research. Using the MYB80 and WUS as targets, the researchers created what they dubbed “Conserved Sequence-guided Repressor Inhibition”, or “CoSRI”.

The name of the protein gives away how it works. This study and previous studies have found that within specific repressors are regions that are conserved across many species. Studies that have removed these conserved regions have resulted in the types of defects discussed above due to the unrestrained transcription of the genes targeted by the subject transcription factors. When the conserved sequence is reinstated, repression of transcription is reinstated and phenotypes return to match the wild-type plants.

Fig 1a from the article. The figure demonstrates how the CoSRI is targeted to the conserved section of the repressor gene and the section of the CoSRI interfering with the binding of the corepressor to the repressor.

So the researchers targeted these conserved sequences as being the sites to direct their newly constructed proteins, thereby building in some specificity to which repressors are targeted. Conjugated to this new protein is a repression-motif interacting region, a section of protein which interacts with the DNA binding section of the transcription factor, stopping the corepressors from interacting with the DNA binding domain of the transcript factor.

Testing the Constructs

Genes for the CoSRI proteins designed specific to the MYB80 and WUS transcription factors were transformed into Arabidopsis thaliana separately.

When the MYB80-specific CoSRI was introduced, the same silique abortion and male sterility were observed. Sterile lines analysed showed that the transcript levels of the CoSRI were at the same levels of greater than the levels of the endogenous transcription factors. Further, transcripts of the genes targeted by the endogenous genes were reduced in sterile lines.

Similarly, CoSRI genes specifically targeting the WUS transcription factor was transformed into A. thaliana, again resulting in altered phenotypes, specifically defective shoot meristems resulting in poor growth and failure of silique elongation leading to loss of fertility.

In short, the researchers had developed a synthetic construct that competed with corepressors for the ability to bind to the repression-binding motif of the transcription factor and blocked the active repression of gene transcription normally mediated by the transcription factors.

Restarting the Repression

Being able to stop the repression of gene transcription is great, but what about reversing it? The example of such a use given in the paper is forcing male sterility in, say, a hybrid plant species, but then being able to reverse the sterility later.

To achieve this, the researchers created what they termed a restorer construct which combined the first 188 amino acids at the N-terminus of the corepressor (the part required to repress transcription by the transcription factor) with a conserved region of the target transcript factor. The result is a protein that is directed to, in one example, the MYB80 transcription factor that carries with it the necessary part of the corepressor to repress the workings of the target transcription factor and restoring the wild-type phenotype.

Figure 1b from the article. The figure depicts the interaction of the CoSRI with the repressor to block corepressors from the repression binding site of the transcription factor, and the use of the restoring CoSRI to reverse the sterility caused by the first CoSRI.

The researchers tested whether the new construct interfered with the transcription levels of any other genes or the transcript levels of the MBY80 targets in wild-type A. thaliana that had not been previously transformed with a CoSRI, finding no interference.

In lines that had both the CoSRI and the restorer construct, sterility was reversed when the transcript levels of the restorer were similar to that of the CoSRI. Fertility was only partially restored or not restored at all when the restorer construct levels were significantly lower than their competition.

Conclusion

This new technique appears to provide an indirect and reversible method of altering the transcription of specific genes. Being able to stop and restart transcription factors for specific genes could be invaluable to plant (and other) research and the technique could find its way in the the toolbox of genetic engineering technology that is exploding at the moment.

The technology could also open up the possibility of being able to alter plant characteristics to suit seasonal requirements, particularly if we develop the ability to not just knockdown and re-initiate gene transcription but also to carefully calibrate gene transcript levels.

 

 

CRISPR Efficiency and Specificity in Arabidopsis

The CRISPR/Cas9 endonuclease system is likely to be the restriction enzyme currently in most widespread use. Its ease of manipulation to target different nucleotide sequences has made it valuable to systems, transgenic and synthetic biology, allowing researchers the ability to observe the effects of manipulating specific genes by knocking them out, down or up regulating them or for adding new genes into genomes.

Related to our recent post about how we identify genes related to traits, many traits that we research and possibly seek to modify, particularly in crop species, are usually controlled by more than one gene. Therefore, studying the effects of altering a particular phenotype characteristic requires the ability to target multiple genes in the one organism.

A recent PLoS One research article looked at the use of CRISPR/Cas9 to target multiple sites in one transformation event in Arabidopsis thaliana. Particularly, they looked at its efficiency transforming a number of genes and also assessed the rate of off-target modifications made.

The Study

The researchers targeted 7 genes from the GOLVEN (GLV/RGF/CLEL) gene family. The proteins produced by genes belonging to this family are important regulators of root stem cell development. In the discussion section of the paper the researchers also mention that the targeting of these genes combined with their selection of guide RNAs resulted in a diversity of DNA sequences being targeted and a significant number of identified potential off-target sites that may also be effected by editing. Therefore, they believe that their results targeting members of this family of genes will be representative of multi-site targeting in Arabidopsis generally.

For each of the seven genes two restriction sites were targeted. gRNA units targeting these sites were synthesised into clusters of 4 and the clusters containing all 14 of the gRNAs were then assembled and cloned into a  binary vector. Wild-type A. thaliana were transformed using Agrobacterium tumefacians via the floral dip method.

The Experiment

Six T1 plants generated from the transformed wild-type plants were screened for editing by sequencing one of the targeted genes (GLV1). Only one of the six plants screened showed a transformation at this location.

The leaves of 48 T2 plants generated from this one transformed T1 plant were sampled in a pool. The pooled DNA was resequenced and the reads mapped against the TAIR10 reference genome.

The criteria the researchers set in assessing whether the targeted genes had been edited was to see whether there were any insertion deletion (indel) events at or near three base pairs upstream of the PAM sequence, noting that while large deletions may not be mappable such large deletions are likely to be infrequent (based on their previous work and experience).

The Results

Editing Efficiency

Using the above criteria and assessing the pooled reads of each of the 14 targets, two of the targets showed no transformation (and from the table below, appear to avoided resequencing altogether. The researchers opined that in their experience such events occur in a similar frequency in single restriction events). Efficiency of editing in the 12 successfully transformed targets ranged from 33% to 92%.

Table 1 from article.

Editing specificity

Being able to target multiple genes linked to a specific trait for mutation is great, but study results can be effected by the endonuclease causing mutations at other sites in the plant DNA. To assess the risk of this occurring the researchers used CRISPRP, a tool that can be used to find highly specific CRISPR sites and allows researchers to assess likely off-target sites within a genome for a particular gRNA. Assessing each gRNA with the tool and limiting the results to the top 20 possible off-target sites for each gRNA sequence, 178 possible off-target sites were identified and were covered in the sequencing data.

Analysing the near 43,500 reads that covered the identified potential off-target sites, the researchers found no indel events attributable to the endonuclease (presumably using the same criteria used to assess whether targeted sites were edited).

Thus, it appears that stacking gRNAs and targeting multiple genes at one time is still highly specific in Arabidopsis.

To check for any other possible off-target events that were not covered in the identified sequences the researchers altered the filters applied to the possible indel sets and obtained a further 4 potential sites that had an allele frequency with with the gRNA of over 3%. Screening these sites in both the wild and the transgenic lines showed a number indel events in both lines, suggesting, say the researchers, that the identified locations are susceptible to deletion events.

Finally, the researchers looked for possible translocations resulting from multiple editing events occurring at once. To do this, they looked for reads of targeted editing events that were contiguous with other editing events (see figure below) and found two independent events, demonstrating that such events can happen but at a very low frequency in Arabidopsis.

Figure 2 from article. The sequences in part A of the figure are target sequences of three gRNA. Part B shows the sequence of the translocation of one of the sequences categorised by the concatenation of the target sequences.

The Discussion

The study suggests that using CRISPR to target multiple genes can result in a targeted and precise gene mutation. However, this is limited to Arabidopsis manipulation and these results do not necessarily extend to other plants. That said, Arabidopsis is widely used model plant for research and the findings here may assist researchers looking to manipulate arrays of genes at the same time.

The low transformation frequency of an admittedly small sample size is a little concerning when compared to high efficiencies of transformation generally seen when CRISPR is used. The researchers point out that this may be due to genes targeted being essential or late embryogensis related. But it would be great to see a replication of the study using other phenotype-related loci to confirm whether there is any transformation effect caused by the genes targeted.

Overall, the results of the study suggest that CRISPR/Cas9 editing continues to find new and useful application and may be of use to those tinkering with plant traits or synthesising new ones.

We Love Open Science!

The Legume Laboratory is delighted to read that one of the scientific journals it sources a significant number of articles from to report on is going to be unleashed from the shackles of restricted access in 2016.

The Plant Biotechnology Journal announced recently that it is going to throw open the journal to free access and distribution, overcoming the frustration of being unable to link to the full article in the pieces written here.

Free and open access to scientific knowledge is at the core of the ideals of the Legume Laboratory and the underpinning principle we believe will improve the lives of people in all parts of the world.

Thank you Wiley Publishing!