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.
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.
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.
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.