Identification and characterization of <i>Tomato spotted wilt orthotospovirus</i> pro-viral factors as a source for durable resistance

Tomato spotted wilt orthotospovirus (TSWV) and related tospoviruses are plant pathogenic viruses that cause extensive crop losses worldwide. Unfortunately, dominant resistance loci against TSWV appear to be (1) scarce, (2) absent in many plant species, and (3) rapidly overcome by resistance breaking isolates of the virus. Therefore, there is an urgent need for growers and our society to discover alternative forms of TSWV resistance. One approach in achieving this is to remove or mutagenize host factors that are essential in the viral infection cycle, which in turn, can convert an otherwise susceptibility host into a non-host for TSWV. Such a pro-viral host factor is also referred to as susceptibility (S)-gene and, hence, applied mutant alleles of S-genes can render durable resistance to TSWV in crops. To identify S-genes for TSWV, a detailed understanding is needed of the host factors hijacked by the virus during its infection cycle. The research presented in this PhD thesis describes different efforts to identify and characterize novel host factors required for the infection cycle of TSWV in order to better understand the molecular interaction between the plant and the virus. These findings can ultimately be applied as a resource of recessive viral resistance in breeding programs. In total, four methods are here presented by which host factors were identified and their role during viral infection in plants was investigated. The pros and cons of the methods applied are discussed together with the applicability of the identified host factors as a novel source for viral resistance

An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in <i>Nicotiana benthamiana</i>

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development

The protein modifier SUMO is critical for integrity of the Arabidopsis shoot apex at warm ambient temperatures

SUMO is a protein modification whose conjugate levels peak during acute heat stress. Here, we found that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of 28 °C. Thermo-lethality at 28 °C was seen in sumo1/2 knockdown mutants but not in 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 in 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 also dependent on the SUMO ligase SIZ1. Thermo-resilience at 28 °C 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 from 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 °C but not 22 °C. We thus describe a novel SUMO-dependent phenotype

Identification of Tomato Proteins That Interact With Replication Initiator Protein (Rep) of the Geminivirus TYLCV

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 EWS-like 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