Host Induced Gene Silencing (HIGS) is a type of RNA interference (RNAi) which has uses in disease resistance. RNAi involves inhibiting the expression of mRNA post transcription by binding complementary micro RNA (miRNA) or small interfering RNA (siRNA) to the transcripts. This can be used in resistance by developing transgenes that can produce siRNAs able to silence essential genes for the pathogen’s survival. HIGS requires genetic modification to insert fungal/viral RNAi cassettes into the host which when infected, the siRNA/microRNA passes into the pathogen and stops the mRNA expression. These DNA cassettes contain a plant promoter, the partial fungal gene in both sense and antisense directions separated by linker and finally a terminator. This cassette is expressed in form of a hairpin in the plant, forming a double-strand RNA (dsRNA). These dsRNA triggers the RNAi machinery in the host and fungus. SiRNAs passes into the pathogen to silence gene expression.
To study the methods which enable the hairpin RNA to pass from the host into the pathogen, a library of mutant Arabidopsis has been created. These mutations are all in genes which code for SNARE proteins – key components of vesicular trafficking which might be critical in the HIGS construct passing from host to pathogen. The SNARE T-DNA mutant Arabidopsis lines obtained from NASC were segregating seeds, a mix of homozygous, heterozygous and wild type plants which needed to be grown and have their genotype tested to select plants which were homozygous for the insertion. This involved growing each line and testing extracted DNA from each plant and using PCR to diagnose the zygosity of each plant. With two primer pairs it is possible to establish their genotype.
When homozygous lines were obtained, these were tested against Fusarium graminearum in a detached leaf assay, which involved cutting leaves, wounding their adaxial surface and placing the petiole in an agar plate, before inoculating the wound with F. graminearum and the mycotoxin Deoxynivalenol (DON). Overall this assay took 4 days to give results- photographs which could then have lesion size measured using imageJ and compared to see if any of the homozygous lines were more susceptible to F. graminearum. This step is important as determine whether any of the mutant lines have increased resistance or susceptibility to the fungal infection before RNAi cassettes are added.
Once the susceptibility had been assessed, the mutant lines needed to be crossed into a different background line. This is because F. graminearum inoculation in Arabidopsis floral tissue is successful in ecotypes presenting the erecta mutation. This sexual crossing involved emasculating the recipient – in this case Colombia erecta Arabidopsis and putting pollen from one of the lines onto the stigma. This technique is extremely hard as Arabidopsis is such a small plant with very fragile flower parts that are easy to break. In addition to this, the crossing needs to happen at the right stage of maturity for the plants. If crossing happens too early, the stigma is underdeveloped and won’t take the pollen properly, whereas if the plants are crossed too late, the plant will already have self-pollinated and the crossing will not work.
Finally, I have done some work inoculating and assessing wheat which has been bombarded and modified to include two different HIGS constructs. Wheat inoculations have happened both at the coleoptile stage and at anthesis of fully grown wheat. The coleoptile assay uses a small piece of filter paper, rolled and placed in a cut filter tip before being soaked with a spore suspension and put over the seedling. After 7 days the plant has grown through the filter tip and has an infected ring of tissue under the filter paper. This assay is very quick and takes up little space meaning it is a high throughput experiment. The other wheat inoculation experiment involved point inoculating spikelets 13 and 14 counting from bottom to top of the flowering wheat ear. The progress of the infection is monitored by eye every 3 days to quantify the susceptibility of the different lines. The results of both experiments could then be statistically analysed and any difference between the infection of a coleoptile or a fully-grown plant could be seen.
Overall my working at Rothamsted Research has been an extremely rewarding experience and introduction into plant pathology. My practical skills have been immeasurably improved by having the chance to work full time in the different labs and growth rooms, as well as my presentational skills and meeting skills. I am extremely grateful to Professor Kim Hammond-Kosack for allowing me to come and work as part of her team, and Dr Ana Machado for all her help and guidance through my time here.
Michael Grimwade-Mann
Bangor University
