Geminiviruses are plant-infecting DNA viruses that reshape the intracellular environment of their host in order to create favorable conditions for viral replication and propagation. Viral manipulation is largely mediated via interactions between viral and host proteins. Identification of this protein network helps us to understand how these viruses manipulate their host and therefore provides us potentially with novel leads for resistance against this class of pathogens, as genetic variation in the corresponding plant genes could subvert viral manipulation. Different studies have already yielded a list of host proteins that interact with one of the geminiviral proteins. Here, we use affinity purification followed by mass spectrometry (AP-MS) to further expand this list of interacting proteins, focusing on an important host (tomato) and the Replication initiator protein (Rep, AL1, C1) from Tomato yellow leaf curl virus (TYLCV). Rep is the only geminiviral protein proven to be essential for geminiviral replication and it forms an integral part of viral replisomes, a protein complex that consists of plant and viral proteins that allows for viral DNA replication. Using AP-MS, fifty-four 'high confidence' tomato proteins were identified that specifically co-purified with Rep. For two of them, an unknown EWSlike RNA-binding protein (called Geminivirus Rep interacting EWS-like protein 1 or GRIEP1) and an isoform of the THO complex subunit 4A (ALY1), we were able to confirm this interaction with Rep in planta using a second method, bimolecular fluorescence complementation (BiFC). The THO subunit 4 is part of the THO/TREX (TRanscription-EXport) complex, which controls RNA splicing and nuclear export of mRNA to the cytoplasm and is also connected to plant disease resistance. This work represents the first step towards characterization of novel host factors with a putative role in the life cycle of TYLCV and possibly other geminiviruses.

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus‐induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants’ performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative‐stranded TSWV and the positive‐stranded potato virus X.

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis – a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype.