Regulation Of Potato Immunity Against Phytophthora Infestans By Ubiquitin-Proteasome E3 Ligases

Late blight, caused by the oomycete Phytophthora infestans, is the most significant threat to potato production. P. infestans delivers pathogenicity proteins into plant cells to suppress immunity, including effectors containing the conserved amino acid motif ‘RXLR’. Plants can detect pathogens and swiftly activate two branches of immune response: Pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI) and effector triggered immunity(ETI). PTI and ETI involve transcriptional changes and de novo production of defense-associated proteins involved in resistance. PTI and ETI responses are regulated by post-translational modifications(PTMs). PTMs are central to protein stability and regulate rapid alterations in protein activity, allowing cells to respond flexibly to pathogens. Ubiquitination is a critical PTM that regulates immunity. Increasing evidence has revealed that PTI and ETI responses require 26 S proteasome-mediated protein degradation following ubiquitination by ubiquitin E3 ligases that act as either positive or negative regulators. In this work, three ubiquitin E3 ligases were characterized: two positive regulators of immunity, PUB17 and Ubox-kinase(UBK), and a negative regulator, POB1. PUB17 and UBK belong to the Ubox class of E3 ligases, whereas POB1 is a BTB/BACK-containing protein that is part of a CRL E3 ligase complex.Work in Chapter 3 shows that potato(St) PUB17 positively regulates immunity in the host nucleus, promoting specific immune pathways triggered by P. infestans. In vitro purified St PUB17 possesses E3 ligase activity. Silencing of PUB17 in potato or N. benthamiana enhances late blight disease, revealing that PUB17 contributes to basal defences. PUB17 is required for programmed cell death(PCD) triggered by CF4 and Avr4 co-expression, as demonstrated by silencing PUB17 in potato, tobacco and N. benthamiana. However, it is not involved in PCD triggered by the P. infestans PAMP protein INF1, or by co-expression of R3 a and Avr3 a. PUB17 is thus not generally associated with pathogen-triggered PCD, but specifically with certain PCD events. Its involvement in CF4-mediated PCD implicates a PUB17-dependent immune pathway triggered by unknown P. infestans elicitors.PUB17 was required for early activation of PTI following treatment with the bacterial PAMP flg22. Early PTI transcriptional responses were attenuated by silencing PUB17. To date, PUBs have not been shown to positively regulate PTI. However, as it is not associated with PCD following INF1 perception, it is not required for all PTI pathways. PUB17 localizes to the plant nucleus. Transient overexpression of a dominant-negative St PUB17 VIVI mutant, which retained nuclear localization, suppressed CF4-mediated PCD and enhanced P. infestans colonization. Exclusion of the St PUB17 VIVI mutant from the nucleus using a nuclear export signal abolished its dominant-negative activity, demonstrating that St PUB17 functions in the nucleus. This suggests that key PUB17 substrates for ubiquitination reside in the nucleus.To investigate how PUB17 regulates plant immunity against P. infestans, identification of its protein interactors in the plant nucleus is critical. PUB17 was found to interact with POB1 by yeast-2-hybrid(Y2H) analysis and the interaction was confirmed in planta. Work in Chapter 4 shows that, surprisingly, transient silencing of POB1 in N. benthamiana increased resistance to P. infestans, whereas transient overexpression of POB1 enhanced P. infestans infection. In addition, overexpression of POB1 suppressed PCD activated by Cf4 perception of Avr4, suggesting that POB1 negatively regulates basal immunity and PCD.POB1 was localized in the nucleus. Exclusion of POB1 from the nucleus abolished its suppression of immunity and the PCD activated by Cf4/Avr4, indicating the nuclear localization is a prerequisite for its regulatory role. YFP florescence of co-expressed YC-tagged PUB17 and YN-tagged POB1 was nucleus-focused, demonstrating that their association occurs at this site. Fluorescence was only detected following treatment with the proteasome inhibitor MG132, indicating that interaction leads to degradation of one or both proteins. Co-expression of m RFP-POB1 with GFP-PUB17 showed a high level of m RFP-POB1 fluorescence in the nucleus but the florescence of GFP-PUB17 was significantly reduced. GFP-PUB17 fluorescence was restored with MG132. This indicated that POB1 negatively regulates immunity by promoting the degradation of PUB17 in the host nucleus.Work in Chapter 5 shows that stable overexpression of P. infestans RXLR effector Pi SFI3 enhanced P. infestans colonization and suppressed flg22-mediated induction of PTI marker genes, suggesting that Pi SFI3 suppresses early PTI response in potato. Y2 H analyses revealed that Pi SFI3 interacts with the ubiquitin E3 ligase UBK. Both proteins localize in the nucleus with a ring around the nucleolus, indicating their interaction likely occurs there. Similar to PUB17, silencing UBK in N. benthamiana by VIGS, or RNAi in potato both enhanced P. infestans colonization and suppressed flg22-induce early PTI marker gene transcription, showing that UBK positively regulates defence. Unlike PUB17, UBK is not involved in Cf4/Avr4-triggered cell death. Also it does not regulate INF1-mediated cell death. Consistent with UBK activity, Pi SFI3 failed to suppress PCD triggered by Cf4/Avr4 or INF1. This indicates that UBK is involved in activation of a PTI pathway triggered by flg22.The three signal pathways triggered by perception of P. infestans PAMP INF1, Cladosporium fulvum effector Avr4 or the bacterial PAMP flg22 require distinct MAPK signal transduction pathways. Present results show that the signaling triggered by flg22 is also positively regulated by the E3 ligases PUB17 and UBK, and Cf4/Avr4-mediated PCD is positively regulated by PUB17 and negatively regulated by POB1. As the two E3 ligases that positively regulate flg22-mediated responses differentially regulate Cf4-mediated PCD, this suggests that they act on different substrates. Present results also revealed that E3 ubiquitin ligases are targets for pathogen effectors which have evolved to inhibit or manipulate their activity. Future work will identify the substrates of the ubiquitin E3 ligases involved in regulation of each signal pathway to understand the mechanisms by which ubiquitination controls defence. In addition, it will be important to explore how pathogen effectors manipulate ubiquitination components to suppress plant immunity.