Objective 1: Optimize conditions for TurboID-based IP in order to identify effector targets.
Using plants co-expressing 35S::PopP2-TurboID-V5 and PAT2::RRS1-HA followed by biotin treatment at a gradient concentration (30-200 μM) for 1 h, biotinylation of RRS1 by PopP2-TurboID-V5 was saturated at 50 μM of biotin, which was selected as the optimal biotin concentration. During the optimization of the labelling duration, I noticed biotinylation efficiency is mainly determined by protein abundance rather than labelling time as biotinylation catalyzed by NLS-GFP-TurboID-V5 at high protein abundance for 30 min exceeded that caused by PopP2-TurboID-V5 at medium abundance for 8 h. Therefore, the labelling time for all my PL-MS was not set and decided based on bait protein abundance. 35S promoter was selected over estradiol-inducing promoter since PopP2 proteins were not detectable when the encoding gene was driven by the latter promoter.
Upon establishing the conditions, Arabidopsis plants overexpressing AvrRps4-FLAG-TurboID and PopP2-FLAG-TurboID were used for TurboID-based PL-MS. WRKY transcription factors, the known targets of AvrRps4 and PopP2, were identified in MS, which proves the feasibility of TurboID-based PL-MS for identifying effector targets. Novel potential interactors of AvrRps4 and PopP2, such as splicing factors and other types of transcription factors, were identified, and the genuineness of which is being tested.
Objective 2: Isolate host targets of A. candida CCG effectors using TurboID-based PL-MS.
To identify the host targets of the 6 A. candida CCG effectors, I generated Arabidopsis plants independently overexpressing the TurboID-V5 tagged CCG effectors. Unfortunately, the CCG effector proteins are below detection limit in all transgenic lines. As a result, the isolation of host targets of the CCG effectors was halted.
Objective 3: Identify pathogen effectors that interact with host targets TCP14 or NLR-ID CHS3 during an authentic in vivo interaction, using TurboID-based PL-MS.
To test whether the method is able to isolate host protein interacting-effectors during infection, Arabidopsis transgenic lines overexpressing TCP14-TurboID-V5 were infected with pathogens P. syringae and Hpa, then subjected for TurboID-based PL-MS. Known host interactors of TCP14 such as TCP8, TCP21 and TCP23 were identified, confirming the feasibility of TurboID-based PL-MS in isolating interactors of host proteins. Only one P. syringae effector, namely AvrE1, but no Hpa effectors were identified. AvrE1 is unlikely the genuine interacting effector of TCP14 as they localize in different subcellular compartments of plant cells. CHS3 carries an integrated Lim domain, which presumably bind the cognate effectors of CHS3. When transgenic lines overexpressing Lim domian-TurboID-V5 were infected with A. candica race Em2 and used for TurboID-based PL-MS to identify the cognate effectors of CHS3, no A. candica effectors were identified. BSL1 and NbVAMP72 of N. benthamiana are targeted by five and seven P. infestans effectors, respectively. When BSL1-TurboID-V5 and NbVAMP72-TurboID-V5 were transiently overexpressed in N. benthamiana followed by TurboID-based PL-MS, no P. infestans effectors were isolated. Collectively, it suggests TurboID-based PL-MS is unable to identify interacting effectors during effectors.
