Hpa pathogenicity

The findings of Fabro et al. (2011) were that HaRxL21 was shown to enhance susceptibil- ity toHpaisolate Noco2. Since the Noks1 isolate was derived from an oospore of Noco2 (Rehmany et al., 2005), the findings here are in agreement with Fabro et al. (2011), in that when constitutively expressedin planta, HaRxL21 is causing enhanced susceptibil- ity toHpaisolate Noks1. Interestingly, this enhanced susceptibility was not found to be isolate specific and enhanced susceptibility was also observed to the isolate Maks9. In both cases, HaRxL21a did not support enhanced growth of Hpa, it may be hypothesised that this is because these plants commonly exhibit the smallest growth phenotype (Figure

3.1). When spraying 14 day oldA. thalianaplants withHpa, it is usual for two true leaves have formed in addition to the cotyledons. The stunted growth phenotype of HaRxL21a results in a smaller leaf surface area for spraying of spores, therefore causing a lower spo- rangiophore count after infection, meaning that scoring ofHpaphenotype in HaRxL21a may show comparable susceptibility.

When theHpaisolate Emco5 was sprayed onto HaRxL21a,b and c, with F1/3, Col-0 and Col-0 GUS controls, a very small number of sporangiophores per seedling were observed across all theA. thalianalines tested which were in a Col-0 background. This was unex- pected because Emco5 has been observed to be virulent on Col-0 seedlings (for example in in Yoshioka et al. (2001)). However, it has been shown that Col-0 exhibits developmen- tally mediated resistance to Emco5, wheras theA. thaliana ecotype Wassilewskija does not (McDowell et al., 2005). Therefore the findings here are consistent with this; the only line which does not show a resistance phenotype to Emco5 was Wassilewskija (ws-eds). In the context of this knowledge, this experiment was therefore asking whether the pres- ence of HaRxL21 compromises the developmentally mediated resistance displayed by Col-0 to Emco5, which was not found to be the case. To determine whether HaRxL21 ex- pression enhances virulence to theHpaisolate Emco5 prior to resistance development, 10 day old HaRxL21-expressing seedlings (too young to exhibit resistance) could be infected with Emco5 and virulence quantified.

3.8.3

Phytophthora infestanspathogenicity

It has been shown here thatin plantaexpression of HaRxL21 enhances growth ofHpaand

B. cinerea, and it has been previously shown that HaRxL21 enhancesP. syringaegrowth (Fabro et al., 2011). It was therefore expected that growth of a pathogen related toHpa, the oomycetePi, would also be enhanced by the presence of HaRxL21.

A. tumefaciensmediated transient expression of HaRxL21 inNbwas used to test whether the presence of this effector enhanced growth of Pi. The effector delivery method used was the same as in Bos et al. (2010) to study function of the Pi effector AVR3a. No difference was observed betweenPigrowth in the presence of HaRxL21 compared to an empty vector control. However, imaging of the biotrophic leading edge of infection using tdTomato RFP was found to be an effective way of monitoringPigrowth compared with observing lesion size in white light. This method will have use in future studies of effector function from bothPiandHpa.

may be due to the effector repertoire of Pi and a redundancy in host targets between effectors fromPiandHpa. Effectors fromHpaandP. syringaehave been shown to share common targets (Mukhtar et al., 2011) so it is likely that more closely related oomycete pathogens may share host targets. It is possible that the susceptibility advantage provided by HaRxL21 toHpamay be pathogen specific, as unlike the obligate biotrophHpa, there is a necrotrophic phase to the Pi infection cycle which may not benefit from the action of HaRxL21. Another explanation may be host specificity, as HaRxL21 may target A. thaliana proteins which are not present in Nb or do not share similar enough sequence homology for HaRxL21 alteration of their function to be conserved across host species.

3.8.4

Botrytis cinereapathogenicity

SA and JA-dependent responses (which plants utilize to defend themselves against biotrophs and necrotrophs respectively) appear to be antagonistic (Glazebrook, 2001) and it has been suggested that pathogens might modulate the balance between SA and JA responses in their favour (Grant and Jones, 2009). This could be useful toHpa, an obligate biotroph, if it is able to remove of this repression of early JA genes and therefore encourage the plant to concentrate its resources in defending itself against necrotrophic pathogens such asBotrytis cinerea(B. cinerea). It might therefore be expected that if HaRxL21 altersA. thalianasusceptibility toB. cinerea, it will be in the direction of reduced susceptibility as the plant is ‘primed’ to defend itself against necrotrophic pathogens due to manipulation of the antagonistic relationship between the SA and JA responses.

A potential mechanism for this reduction in susceptibility can be defined by studying the model for JA gene regulation by Pauwels et al. (2010), which suggests that early JA genes are repressed by the recruitment of Novel Interactor of Jaz (NINJA) which in turn recruits TPL through direct binding via the EAR motif, therefore bringing about transcriptional repression by histone modification (Pauwels et al., 2010). One of the interacting protein targets of HaRxL21 was found by Mukhtar et al. (2011) to be TPL and the amino acid sequence of HaRxL21 also contains an EAR motif, akin to NINJA. One possible hypoth- esis, therefore is that HaRxL21 may therefore interfere with this pathway, possibly by titrating TPL away from the complex and therefore removing repression of JA responsive genes.

TheB. cinereaphenotype observed did not agree with this hypothesis, as we would have expected a reduction in growth of this pathogen. This result is unusual as we might not expect an effector protein from an obligately biotrophic oomycete pathogen to perturb defense against a necrotroph such asB. cinerea.

One of the aims here was to establish whetherA. tumefaciensmediated transient expres- sion inNb could be used to screen for susceptibility advantage byHpaeffectors, in par- ticular HaRxL21. This system would allow for rapid screening of mutated versions of the effector to determine functionality of different domains. However, the susceptibility advantage conferred toB. cinereainA. thalianawas not replicated inNb. These results, together with the result that transient expression of HaRxL21 does not enhance suscep- tibility to Pi, suggest that maybe the downstream targets of HaRxL21 are not present in

Nb. This could be investigated further by yeast-2-hybrid (Y2H); determining whether the interacting partners of HaRxL21 have relatives inNbwhich HaRxL21 is able to interact with.

The results obtained in figure 3.12 suggest that expression vector plays a role in pathogenic- ity effects of HaRxL21. This may be because the presence of the HA tag is interfering with the effector function, for example localisation, or binding of protein or DNA targets. Using the pGRAB vector for transient expression of HaRxL21 inNicotiana benthamiana

and comparing it to either empty pGRAB or GFP in the pGRAB vector might yield results more concurrent with the enhanced B. cinerea phenotype observed duringin planta ex- pression of HaRxL inA. thaliana. However the down-side of using pGRAB for transient expression is that it would not allow for detection of protein levels by western blotting (ex- cept by specific antibodies to the effector), to ensure that expression continues throughout the infection time course.

3.8.5

Pseudomonas syringaepathogenicity

Using the EDV system (Sohn et al., 2007), it was shown that HaRxL21 delivery enhances growth ofPst luxcompared toPst luxexpressing AvrRPS4-AAAA control, but not com- pared toPst luxalone, 96 hours post infection. HoweverPst luxalone is not a represen- tative control in this case, as it it likely that the process of replicating the EDV6 vector may slow bacterial replication compared to Pst lux alone. The variability in this screen is high however, due to the low number of replicates possible within plant growth and time constraints. Fewer time points (for example taking samples 24 h post infection is not necessary) would enable greater replication and increase confidence in these results. It is also possible that within the effector arsenal ofP. syringae, there are effectors which function in a similar manner to HaRxL21. LikeHpa,P. syringaeis a biotrophic pathogen and therefore similar host targets would benefit both pathogens. The soil bacteriumPseu- domonas flurescenshas been engineered to express the type III secretion machinery from

the virulence of HaRxL21 to be assayed without possible interference or overlap fromP. syringaeeffectors, and should be used for any future screens of this kind.