Recognition of the flax rust fungal effector, AvrM, by the M flax rust resistance protein

Author: Thi Danh Vu

Vu, Thi Danh, 2019 Recognition of the flax rust fungal effector, AvrM, by the M flax rust resistance protein, Flinders University, College of Medicine and Public Health

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Plants that live in the natural environment must deal with many stressful factors, none more so than disease causing micro-organisms. Crop losses affect the incomes of many families whose livelihoods depend on agricultural production and they also pose a major threat to food security worldwide. In order to contribute to our understanding of the plant pathogen interface, the research presented here has focussed on the interaction between the flax rust fungal effector (AvrM) and the flax rust resistance protein M. Resistance proteins, like M, have evolved to recognise the presence of pathogen effectors that are secreted into plant cells. In this research, we have used two flax rust effector proteins, AvrM and avrM, in which the AvrM effector-containing strains of flax rust elicits a disease resistance response when inoculated onto flax plants that contain the M flax rust resistance gene. Both AvrM and avrM were isolated from Melampsora lini. The avrM protein, despite sharing 96% similarity to the AvrM protein, does not elicit a response when avrM-containing strains are inoculated in the same way. With the isolation and cloning of the AvrM and avrM effector genes, and the flax rust resistance gene M, this interaction can be uncoupled from its natural plant/pathogen system and the effectors expressed transiently via Agrobacterium infiltration into transgenic tobacco stably transformed with the M gene. That is, when Agrobacterium tumefaciens carrying either the AvrM or avrM genes is infiltrated into transgenic M-containing tobacco plants, a characteristic disease resistance response or hypersensitive cell death response (HR) may be generated in the tobacco tissue dependent on whether the AvrM/M proteins are interacting. We have used this experimental system to investigate the specific amino acid differences between AvrM and avrM that control recognition and activation of the M rust resistance protein. The work to identify mutants in AvrM that can knockdown M resistance protein-induced HR or knock-in that of avrM was part of the PhD project of Motiur Rahman (Rahman, 2016). One limitation of Rahman’s study was that all 19 mutants of both AvrM and avrM were shown the change of HR in planta. However, the expression of these proteins in the tissue was not confirmed by western blot analysis. This was the aim of this study. From the structure of the AvrM and avrM proteins (Ve et al, 2011), a small charged pocket was found at the interface of the AvrM homodimer that was unique to AvrM and not found in the avrM structure. Residues E237, E309 and R313 were non-polymorphic between AvrM and avrM and yet were responsible for this charge difference. Based on this, Rahman (2016) made seven mutant forms of AvrM, namely AvrM E237A, AvrM E309A, AvrM R313A, AvrM E237A+ E309A, AvrM E237A+R313A, AvrM E309A + R313A, AvrM E237A + E309A + R313A, and found that all single mutants and some double mutants were recognised by M, while the AvrM E237A+ E309A double and all triple mutants were not. Rahman (2016) concluded from this, that the charged pocket in the AvrM structure is essential for M recognition. A minor difference between the study of Rahman and this study was that here only the triple mutant, AvrM E237A + E309A + R313A, had the ability to knock down the recognition of M protein, whereas Rahman showed that the double mutant (AvrM E237A+ E309A) was able to do this. Of the 13 residues that were polymorphic between AvrM and avrM, Rahman (2016) made eight single mutants in the avrM effector, and in a number of double, triple, and quadruple combinations. The avrM mutants were created by changing the identity of amino acid in avrM to that of AvrM. This was done in order to see what polymorphic residues were important in M recognition. Work presented here, showed that M recognition was partially restored in the avrM R170K + S179L and avrM R170K + T247I double mutants. To further investigate the role of key residues involved in M recognition, Rahman (2016) made the reciprocal changes to those discussed above in AvrM, to see if M recognition could be knockout. For four mutants of polymorphic residue in the AvrM effector: AvrM K232R + L241S, AvrM K232R + I310T, AvrM L241S + I310T and AvrM K232R + L214S + I310T, all were recognised by the M protein to same level as that of AvrM. These changes could not knock down the M recognition. Work presented here showed by diluting the Agro-infiltration of the mutants, (avrM S179L + T247I and avrM R170K + S179L + T247I) had a weaker M-induced HR than AvrM. Further to the work of Rahman (2016), all the effector proteins were shown here to be expressed in tobacco leaves to similar levels as demonstrated by Western blot analysis. Therefore, any difference in HR could not be attributed to differences in protein expression, and rather by the recognition of the effector by the M protein. In a related but parallel study, the effect of light on the strength of the HR was assessed in the M/AvrM interaction assay. The M-AvrM induced HR was inhibited when tobacco leaves were pre-treated with darkness for 72, and 48 hours before infiltration in comparison with that of a 24 hours dark pre-treatment and the control. The effector protein showed equivalent levels of expression in the protein samples extracted from dark treated leaves and was higher than that from the control leaves. This result confirms reports in the literature that light is required for the activation of the R-protein induced HR. A possible explanation for this is discussed.

Keywords: The flax rust fungal effector, AvrM, M flax rust resistance protein

Subject: Biotechnology thesis

Thesis type: Masters
Completed: 2019
School: College of Medicine and Public Health
Supervisor: A/Prof Peter Anderson