3.4.1 Cultivar P55/7 shows resistance to Pa1 while Picasso does not
Roots of Picasso and P55/7 plants were inoculated with Pa1 cysts and left for 8 weeks before the total number of females present on the root systems were counted (Supplementary Table 1). Four plants of each cultivar had three replicates and two independent screens to test their resistance/susceptibility to G. pallida Pa1 (Picasso had 21 out of 24 successful replicates, while P55/7 had 20). Picasso had a large spread of infection scores with one replicate having 54 females present, while another replicate had only 2 females, however the mean infection score for the cultivar was 17. P55/7 had a much narrower range on infection scores with a mean infection score of 0.65 females per replicate (Figure 3.3). The error bars were calculated based on the standard deviation for Picasso (SD=15.11) and P55/7 (SD=1.04) with a p-value <0.001 making the scores statistically significant, Picasso is susceptible to Pa1 while P55/7 is resistant.
Figure 3.3 Graph of Pa1 scores from meristem infection for Picasso and P55/7. Error bars were calculated using the standard deviation, Picasso (SD=15.11) and P55/7 (SD=1.04), and a p-value of <0.001.
3.4.2 dRenSeq identifies R genes present in the susceptible parent Picasso
To evaluate if the resistance phenotype in the resistant clone P55/7 and segregating within the F1 progeny could be associated with an already characterised R gene, diagnostic RenSeq (dRenSeq) analysis was conducted. RenSeq enriched paired-end reads were mapped against a panel of known functional NB-LRRs including the nematode resistance genes Gpa2, Gro1.4, Hero and Mi1.1/1.2 to assess their presence within Picasso and P55/7. For a gene to be designated as ‘present’ within a cultivar the sequence had to match 100% with the reference gene coding sequence. DRenSeq analysis revealed the presence of G. pallida R gene Gpa2, P. infestans R genes R1, R3a and R3b, and the viral PVX R gene Rx in Picasso; all whose presence was previously unknown. In comparison, no previously published functional R genes were revealed to be present in P55/7.
Results in Figure 3.4 give a visual representation as to how the reads mapped to each of the reference genes. As an example, in the case of Gpa2 which is present in Picasso, read depth ranges between 10-100 across the whole length of the gene, with 100% of the gene being covered. By comparison, P55/7 which doesn’t have a functional copy has variable levels of read depth with less than 25% of the gene being covered against the reference sequence. Some genes have sections of sequence similarity (Rpi-blb1, Rpi-R8, Rpi-R9) but have been designated as not present; this is due to the repetitive nature of NB-LRR genes, and the allowance of sequences to locate to 10 separate positions during mapping which can cause peaks to appear where small areas of sequence similarity are identified. Accepting only genes with 100% sequence coverage as present and functional revealed the presence of five functional R genes in susceptible Picasso, while no previously known functional R genes were identified in P55/7, giving a strong indication that H2 is a discrete R gene which has not been previously described and is controlling the observed resistance phenotype.
Interestingly, the form of R3b which is present in Picasso, and has the ability to recognise Avr3b is not the published form of the gene. The identified copy of R3b contains within it
two non-synonymous mutations R3bG1696/G3111 (Armstrong et al, 2018). These
mutations have been verified as correct and present through sequencing and functional testing work undertaken by another student (Strachan et al (submitted)). This type of allele mining which dRenSeq allows, highlights its suitability as a tool for R gene identification and validation.
Figure 3.4 dRenSeq analysis of parent cultivars Picasso (red) and P55/7 (black). Each trace is representative of a known NB-LRR in a given sample. The x-axis displays the coding sequence of the gene and the y-axis is a log10 scale of read coverage. NLRs with full coding sequence representation are in bold.
3.4.2 Transient expression proves presence of functional R genes
dRenSeq analysis identified the presence of five functional R genes in Picasso, while no already characterised R genes were identified in P55/7. To verify these findings, transient expression via vacuum infiltration was undertaken to prove the functionality of the identified genes. Cognate effectors for each resistance (R3a::Avr3aKI (Bos et al., 2009), R3b::Avr3b (Li et al., 2011), Rx::Cp (Bendahmane et al., 1995) and Gpa2::RBP1 (Sacco et al., 2009)) were used as well as negative controls eGFP and Avr2 which should not give a response as R2 is not present (Gilroy et al., 2011), and positive control CRN2 (an effector known to induce cell death) (Haas et al., 2009). Unfortunately R1::Avr1 could not be tested during this experiment, and so the functionality of R1 could not be verified.
R gene-mediated cell death symptoms started to become visible after six days, however results became more pronounced 10 days post infection and so this time point was used to analyse the experiment. Picasso leaves showed varying symptoms of cell death with the Rx-mediated cell death showing complete necrosis across the entire leaf, while the eGFP control showed no signs of cell death or necrosis. Cognate effectors Avr3a, Avr3b and RBP1 showed cell death, while Avr2 did not show signs of necrosis (Figure 3.5). Elicitation of the target R3a, R3b, Rx and Gpa2 R genes proves both the presence and functionality of these genes within susceptible Picasso.
In comparison, transient expression of the same cognate effectors in P55/7 did not elicit any visible cell death response (Figure 3.5). After 10 days of incubation no R gene-mediated symptoms could be visualised and so it can be accepted that the dRenSeq analysis which identified that no currently characterised R genes are present in resistant P55/7 is correct. It could be argued that a lack of a cell death response in P55/7 is due to insufficient or no bacteria being successfully infiltrated into the leaf. However, the positive cell death response which is elicited when CRN2 is infiltrated (Torto et al., 2003) displays that it is a
Using the results from the transient expression experiment confirms the dRenSeq analysis that five characterised R genes are present in Picasso, while none of these are present in P55/7. These results also confirm that Gpa2 (present in variety Picasso) does not control G. pallida pathotype Pa1 in P55/7 and the resistance phenotype is indeed mediated by the H2 R gene rather than a previously characterised R gene.