Tag Archives: wheat

Host-delivered RNAi to control Wheat Leaf Rust

Wheat is a major staple food worldwide. Accordingly, threats to wheat production pose a significant risk to global food production and security. One major threat to wheat production is Wheat Leaf Rust (“WLR”), a disease caused by the fungus Puccinia triticina which has the ability to cause severe yield losses. Of further concern, the application of fungicides to control the disease can lead to the development of reduced sensitivity or resistance to the fungicide, let alone the economic and environmental impacts that fungicide use entails.

Given the serious threat that WLR entails and the problems with using fungacides, genetic studies have been used to identify a number of resistance genes which have been used to breed cultivars with greater resistance to the fungus. However, the use of resistant cultivars in monoculture applies significant pressure on the fungus to evolve and develop new methods of virulence.

The authors of the paper being presented today outline these important problems noting that, when combined with the effects of a changing climate, disease outbreaks are at risk of increasing and further threatening food security.

A Potential Solution?

After discussing the life cycle and infectivity of the fungus, the researchers behind the paper published in the Plant Biotechnology Journal theorised that by using the increasing genetic knowledge about the pathogen, particularly the genes implicated in their virulence factors that enable it to infect wheat plants, it may be possible to target and repress those genes using RNA-interference (RNAi).

We have written about RNAi previously in the context of RNAi sprays as a possible alternative to traditional pesticides, potentially being used in Bioclay-based pesticide and in transgenic plant pathogen resistance.

The researchers outline the possibility of using host-delivered RNAi (“HD-RNAi”), inserting genetic material into the plant that will translate into a double-stranded piece of RNA complementary to one of the virulence genes of the pathogen. When the dsRNA piece is cleaved into small interfering RNA (siRNA) duplexes in the plant and delivered to the pathogen, the siRNAs bind to the transcripts of the complementary virulence gene in the parasite and interfere with their further translation.

Using recent studies that characterised virulence genes of rust fungi, the researchers chose to target the Pt Map-kinase (PtMAPK1) and Pt cyclophilin (PtCYC1) genes, attempting to silence their translation and discover whether effective silencing of these genes will afford any protection to the plants against the fungus.

Results

HD-RNAi constructs targeting each gene were transformed (separately) into immature embryos of a WLR susceptible wheat cultivar via projectile bombardment, creating 14 transgenic To lines harbouring the hp-PtMAPK1RNAi construct and 20 To lines aimed at silencing the PtCYC1 gene. These lines were selfed to produce T1 seeds. Polymerase chain reaction (PCR) was used to verify successful insertion of the constructs and reverse-transcription PCR (RT-PCR) was used to verify transcription of the construct. Furthermore, there was no effect on phenotype seen in the transformed lines.

The transformed lines and a set of controls were then challenged with the Pt urediniospores when they were two weeks old. 12 days after being challenged, the plants were scored on a 0 to 4 scale depending on the size and abundance of uredinia, with 0 being little to no infection and 4 being highly susceptible.  A total of 13 of the transformed lines (6 of the PtMAPK1, 7 of the PtCYC1) showed varying resistance to the fungus compared with the controls. Those lines with the greatest levels of resistance showed symptoms of infection between 1 and 3 days later than the control lines did.

Figure 1 from article showing WLR infection levels in select transgenic lines compared to control (on the left).

To further investigate whether the HD-RNAi were the cause of the difference in susceptibility to the fungus, the researchers looked at the rate of fungal growth within the leaves of the plants by comparing the ratio of fungal DNA to plant DNA between the transgenic lines and the control lines. Analysis showed that the amount of fungal biomass was reduced by nearly 80% in each line compared to the controls. Accordingly, it appears that the HD-RNAi constructs have managed to interfere with the fungus’ ability infect and grow in the plant.

Using the quantification of fungal biomass to group the plants with differing levels of WLR resistance, the connection between the level of resistance and the severity of disease was tested. It was found that there was a close relationship between the two criteria, with the lowest disease severity correlating with the lowest ratio of fungal biomass to plant biomass.

Figure 5 from article. (a) Comparison of fungal and plant biomass within infect control and transgenic wheat lines. (b) Transcript levels of genes targeted by the RNAi construction compared to control. (c) Confocal microscopy of infected cells.

To test whether the HD-RNAi constructs were actually interfering with the targeted fungus genes, quantification of transcript levels of the fungus genes was performed 5 days post infection of both transgenic and control lines. Transcript levels of both target genes was reduced by between 41% and 65% in transgenic lines compared to controls, and the reduction of transcript levels of each particular gene was specific to the transgenic line expressing the HD-RNAi construct targeting that specific gene. Further, the measured transcript levels correlated with the level of disease severity in the plant, demonstrating a link between the reduction in target transcript, level of fungal biomass and disease severity.

Finally, three transgenic lines from each HD-RNAi transformation showing increased disease resistance were selfed to obtain T2 generations of each. PCR and RT-PCR confirmed the presence and translation of the gene construct in that generation and the plants were challenged with Pt urediniospores when they were two weeks old. Similar to the T1 generation, infection scores were lower in the transgenic lines compared to control lines, with a similar delay in onset of symptoms previous described.

The same analysis of fungal biomass and transcript abundance was measured, again with similar findings compared to the T1 generation.

Discussion

The results of the research are promising, and the discussion section of the paper explored the possibility of targeting more than one virulence gene in any one transformation event or targeting different virulence genes as we learn more about the genetics of the fungus.

Further discussion centres on how the HD-RNAi constructs are delivered to the pathogen, a process not fully understood. It is hypothesised that the HD-RNAi transcripts may enter the pathogen via vesicle-mediated transport across the haustorial interface. However, the delivery method requires further research to confirm this and to confirm how inter-kingdom RNA is able to interfere with one another.

Why there was a variation in resistance levels amongst the different transgenic lines was also discussed, with hypothesis such as incomplete knockdown of the target gene, variability of gene expression levels and target gene transcript turnover rates.

Conclusion

The study builds upon our growing knowledge of plant and pathogen genetics, pairing the two with our understanding of RNAi to explore the possibility of controlling a significant and increasing threat to wheat production and avoiding the fiscal and environmental problems that come with increasing fungicide use. The results of the study are promising and should direct further research. This further research may consider whether different pathogen genes can be targeted, whether targeting different combinations of genes will have greater effect, and testing whether the problem of forced evolution of the pathogen to overcome our advances will mean that our crop defence strategies will still require us to employ agricultural practices to reduce the evolutionary pressure our genetic strategies apply.

A great study with possible applicability to a range of pests in other important crops.

Does C4 photosynthesis occur in wheat seeds?

In our humble opinion, the debate about the conclusions drawn in a recent paper regarding the possibility of C4 photosynthesis being active in wheat seeds is one of the more interesting scientific debates raging at present.

The debate started with a paper published by Rangan et al last year which described findings they had made about differential expression of genes implicated in C4 photosynthesis within wheat seeds. These expression levels differed to the gene expression levels in other parts of the plant. We wrote an article describing the research here.

The response to that article (and our post) was that the evidence provided, although novel and interesting, wasn’t enough to justify the conclusion that C4 photosynthesis was in fact occurring within the wheat seed. Noting the criticisms, we wrote an updated piece covering two published responses to the Rangan article.

But the debate hasn’t stopped there…

Back and forth we go

The journal Plant Physiology published a series of letters between the researchers who published the initial research and two researchers with the differing opinion. The series of letters outlines the main points of difference between the propositions.

In defence of the C4 pathway conclusion

The series starts with a defence of the conclusions drawn from the research. Citing the earlier work of Bort et al (1995), a paper which described labeled carbon assimilation differences between leaves and seeds of wheat and barley and which found no significant difference between the location of the assimilated labeled carbon, the defenders argued that the research did not distinguish between carbon assimilated in the glumes covering the seed (the Bort et al paper) and carbon assimilated in the pericarp (the Rangan et al research).

Dissection of Wheat Glume showing glume and grain.

Figure from article showing cross-section of wheat seed, particularly the pericarp.

The distinction between the two asserted is that the Rangan et al research shows that pericarp, as opposed to the glume, demonstrates elevated C4 gene transcription.

Further, it is suggested that the carbon source supplying the pericarp is not from the capture of external CO2 but instead comes from the endosperm capturing respired carbon, the carbon being derived from bicarbonate in the developing seed tissue, and moving outwards to the pericarp for use in the asserted C4 pathway. For this reason it is suggested that the conclusions drawn by Bort et al, that there was no evidence for C4 photosynthesis in the ears of C3 cereals from their labeled-carbon pulse experiment, is a false basis to deny their conclusion as it failed to account for this inside-out carbon delivery.

To the contrary

The first point raised in contradiction to the Rangan et al research repeats the earlier criticism – that although the gene expression profiles reported in the disputed paper are novel, interesting and worthy of further research, they alone are not enough to justify the conclusions made. To be able to make such a conclusion, evidence demonstrating flux of the metabolites through the C4 pathway. Increased expression of the relevant genes is not enough to conclude that the pathway is operating in the seed particularly as all the genes involved in C4 photosynthesis are also expressed in C3 plants.

Although the evidence for the existence of the pathway in wheat seeds is scarce and contradictory, the defenders of the rebuttal suggest that the Bort et al article is evidence that the PEP carboxylase activity assists in intermediate reactions in metabolic pathways other than C4 photosynthesis.

Further, the evidence in the Rangan et al paper suggested that the expression of genes encoding the Rubsico enzyme was minimal. The contention is that, if the increased transcription of C4 genes is relied upon as evidence of increased activity within the pathway, reduced transcription of this vital photosynthetic enzyme must lead to the conclusion that the increase in PEP carboylase activity must be in aid of some pathway other than a photosynthesis pathway. Coupled with this, the low concentration of CO2 in the pericarp, where the Rubisco is located, is contrary to the high concentration of CO2 around Rubsico common in C4 tissues. Accordingly, expending energy to run a C4 pathway when the carbon would flow to Rubsico within the pericarp without the need for the pathway is contrary to expected energy conservation measures.

…and back again

In response to the criticism that evidence of flux through the pathway is lacking, the defenders of the Rangan et al paper suggest that such evidence has already been reported in a 1976 paper in the C3 intermediate barley and the relevant proteins isolated in research reported in 1986. It is argued that their research has showed that C4 specific versions of the genes were expressed in the seeds compared to the C3 versions expressed in the leaves.

In relation to the Rubisco levels, the Rubsico levels reported in the paper were for the whole seed, not just the pericarp and earlier work has shown that Rubsico is specifically expressed in the pericarp with very limited levels in the endosperm. As such, it is argued that the although the levels were rather limited in the seed as a whole, they are in fact present in high concentrations in the pericarp, the site where the C4 pathway is said to concentration CO2 levels.

Returning to the problem of the Bort et al paper, the researchers again suggest that the experiment conducted in that research could not have tested for the possibility of a C4 pathway supplying CO2 to the pericarp from within the seed, after which it would be utilised by Rubsico concentrated in the pericarp. Accordingly, they argue that the conclusions of that study fail to invalidate their conclusions.

As for the possibility that CO2 could more efficiently diffuse to the pericarp, it is contended that if that was the case it would not be concentrated enough at the site of the Rubisco and would therefore all too easily escape the seed. Their contention is that the presence of a C4 pathway in the seed is necessary for adequate CO2 concentration to the pericarp-located Rubisco.

…and one last response

The final of the letters concedes that the labeling of CO2 inside the endosperm may yield different results compared to the labeling study in the Bort et al paper, but cites the 1976 research as using isolated pericarps in their experiment and therefore doesn’t provide appropriate evidence support the C4 pathway inside the endosperm.

In relation to the diffusion versus C4 pathway dispute, it is suggested that any CO2 which is not passed from the endosperm to the pericarp through intermediates in the pathway would diffuse outwards following Fick’s law whether or not it moves through the tissue as malate. If it moved as malate, again the cost of such a pathway would be more than if it moved by simple diffusion.

Conclusion

The last paragraph of the final letter sums up the research as it stands and the debate about the possible conclusions that can be drawn. The research in the Rangan paper provides “an exiting new piece of the puzzle” in the quest to understand how wheat seeds increase carbon gain. It would seem that to put the debate to rest, the flux of the carbon through a C4 pathway must be demonstrated unequivocally along with measurement the location and activity of the required enzymes.

A passionate and reasoned debate over plant science which is wonderful to see.

Researchers put some of the wild back into domesticated wheat — Scientific Inquirer

Kansas State University scientists are part of a breakthrough study in which an international team of researchers has successfully deciphered all 10 billion letters in the genetic code of a wild ancestor of wheat. Their work is published in Science Magazine. “The relative of wheat is called wild emmer, which is one of the founding […]

via Researchers put some of the wild back into domesticated wheat — Scientific Inquirer

Decoding Diversity in the Food System: Wheat and Bread in North America

Philip H. Howard Abstract: Diversity is important for the resilience of food systems, as well as for its own sake. Just how diverse are the systems that produce our food? I explore this question with a focus on wheat and bread and North America, and even more specifically in baking, milling and farming. Although the […]

via Decoding Diversity in the Food System: Wheat and Bread in North America — Philip H. Howard