J, K. | bioRxiv Science

bioRxiv2024

Branched DNA Processing by a Thermostable CAS-Cas4 from Thermococcus onnurineus: Expanding Biochemical Landscape of Nuclease Activity

The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral "spacer" sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5' to 3' exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5' direction, demonstrating structure-specific endonuclease activity.

biochemistry↗

bioRxiv2023

Multiomics-assisted characterization of rice-Yellow Stem Borer interaction provides genomic and mechanistic insights into stem borer tolerance in rice

Yellow stem borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice in India, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate source of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB tolerance at vegetative phase in a highly tolerant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB tolerance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and tolerant lines revealed multiple genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB tolerance. Taken together, our study provides insights on rice-YSB interaction at genomic, transcriptomic and metabolomic level, thereby facilitating the understanding of tolerance mechanism. Importantly, a promising breeding line and markers for YSB tolerance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars. SIGNIFICANCE STATEMENTGlobal rice production is threatened by various pests, among which stem borers pose serious challenges. Hence, understanding the molecular intricacies of rice-stem borer interaction is necessary for effective pest management. Here, we used a multi-omics approach to unravel the mechanisms that might help rice combat yellow stem borer infestation, thus providing insights and scope for developing YSB tolerant rice varieties. To facilitate the latter, we developed markers that co-segregate with tolerance.

plant biology↗