Viral capture to identify susceptibility genes (VICIS) in Brachypodium distachyon, barley and wheat

As a result of the global corona pandemic, everyone has experienced the immediate impact of a viral epidemic on human health. However, it is often underestimated that similar repercussions can arise when crops instead of humans become hosts to pathogens. Up to 40% of global yield losses are attributed to pathogen-induced diseases, resulting in an annual economic cost exceeding US$220 billion. With the world’s population rapidly expanding, it has become unaffordable to lose yields due to plant diseases. Effective management of viral diseases is critical to augment crop productivity per unit area and optimize land use.

This thesis focuses on the introduction of viral resistance in plants. Viruses, with their compact genomes, will exploit plant-specific proteins to establish a successful infection. We call these proviral factors susceptibility factors (S-factors), since their presence ensures a compatible virus-plant interaction. Consequently, by disrupting or suppressing these factors the compatibility between the plant and the virus can be compromised, resulting in viral resistance. RNA plays a central role in the infection process of single-stranded positive sense RNA viruses, functioning both as a genome as well as an mRNA. Therefore, it is not surprising that the vRNA physically interacts with a  myriad of host-derived RNA-binding proteins (RBPs) to facilitate critical steps for successful infection, such as translation, packaging, localization, etc. Identifying the viral RNA-host protein interaction could result in a list of interesting targets for antiviral strategies. In recent years, numerous techniques have been develop to study RNA-protein complexes (RNPs). RIC was the first RNA-centric method to allow the isolation of the mRNA interactome targeting the RNPs poly-A tail. Multiple other techniques such as CARIC, RICK, TRAPP, VIR-CLASP to isolate a compilation of RNPs have been developed since. Recently, new methods, based on organic phase separation, were developed to isolate the whole compendium of RNPs without a selection of certain RNA elements or post-translational modifications. Additionally, instead of targeting a whole set of RNPs, methods to isolate specific RNP complexes also emerged with ChiRP-MS, CHART-MS and RAP-MS. However despite the currently available set of techniques, to our knowledge only a few interactomes of specific RNA species have been identified. For plants no specific RNP isolation protocol was described before. While proven to be successful, we believe that technical limitations, yield, purity and high experimental cost of previous procedures prevent them from being routinely used. We identified the origin of these difficulties and combined existing approaches to overcome these challenges.

We developed a novel methodology termed silica-based acidic phase separation (SAPS)-capture for the purification of specific RNPs. This approach involves a silica-based isolation of RNA and RNPs followed by an organic phase separation to specifically isolate all RNPs. Subsequently, the specific RNP of interest is targeted by the use of antisense biotinylated probes. By including the prepurification of the RNPs before specific RNP isolation, we achieved significant improvements in cost, scalability, and buffer flexibility compared to existing protocols. We validated our method by isolating a well-characterized complex, namely 18S ribosomal RNPs in baker's yeast. Performing the protocol with prior knowledge of the expected outcome enabled us to assess the specificity of the procedure. Following the SAPS-capture procedure, we found that 80% of the significantly enriched proteins were indeed interactors of the ribosomal RNA, confirming the method's effectiveness.

To expand the applicability of the protocol to more challenging samples, such as plant material, we adapted the SAPS portion of the protocol to Arabidopsis thaliana. This involved adjusting the lysis buffer and enhancing the UV cross-linking procedure to efficiently cross-link whole tissue samples. Given our belief in the organism-independent nature of the SAPS output, the successful isolation of a confident plant RNA-binding proteome in A. thaliana encouraged our confidence in the applicability of the whole SAPS-capture procedure to plants.

The in-house developed SAPS-capture was subsequently applied to purify viral RNPs for the identification of S factors involved in Tobacco rattle virus infection (TRV) in Nicotiana benthamiana. Our analysis revealed three viral proteins and an enrichment of nucleotide-binding proteins, serving as positive controls for the protocol. Furthermore, we identified 36 interaction partners of the viral RNA, which we are currently validating. In other words, exploring whether disrupting these genes results in a significant reduction in viral replication. While the initial screening of the T1 generation showed promising results (12 targets w/o TRV/2targets with reduced TRV), the lack of genetic uniformity necessitates further validation in replicate using stable knock-out lines. Given that S factors are commonly regarded as broad-spectrum resistance factors, our findings hold potential for translation to  crops like tomatoes and potatoes, which belong to the same plant family as N. benthamiana and are susceptible to TRV-related viruses.