The Old-Javanese Rāmāyaṇa Kakawin

Indonesi

CRISPR/Cas9 mutagenesis by translocation of Cas9 protein into plant cells via the Agrobacterium type IV secretion system

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNAguided Cas9 endonuclease is usually delivered into plant cells as a DNA construct encoding Cas9 and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations. In addition, DNA constructs can integrate into the host genome, causing mutations and complicating regulatory approval. Instead of DNA, here we deliver Cas9 through the Agrobacterium T4SS, accomplished by fusion of the VirF T4SS translocation peptide to Cas9 (NCas9F). Co-cultivation of Agrobacteria expressing NCas9F with yeast (Saccharomyces cerevisiae) harboring a sgRNA targeting CAN1 showed that NCas9F was translocated via T4SS and induced targeted mutations in the yeast genome. Infiltration of Nicotiana benthamiana leaves with Agrobacteria expressing NCas9F and sgRNA-PHYTOENE DESATURASE (PDS) resulted in targeted modifications at the PDS locus, albeit at a very low rate. In order to increase the mutation frequency NCas9F protein was co-transported with a TDNA encoding sgRNA-PDS1. Next generation sequencing confirmed that this resulted in targeted mutations at the PDS locus with a similar distribution but at a 5-fold lower frequency as the mutations obtained with a T-DNA encoding both Cas9 and sgRNA-PDS1. Similarly, infection with Tobacco rattle virus (TRV) encoding sgRNA-PDS2 combined with NCas9F protein translocation resulted in an equally high frequency of PDS mutations in N. benthamiana compared to T-DNA encoded sgRNA-PDS1 combined with NCas9F protein translocation. Our results revealed that translocation of NCas9F protein via the Agrobacterium T4SS can be used for targeted mutagenesis in host cells instead of the permanent and constitutive expression of Cas9 from a T-DNA.Plant science

Ctr2 Links Copper Homeostasis to Polysaccharide Capsule Formation and Phagocytosis Inhibition in the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is a human opportunistic fungal pathogen responsible for ∼1/3 of HIV/AIDS deaths worldwide. This budding yeast expresses a polysaccharide capsule necessary for virulence. Capsule production inhibits phagocytosis by macrophages. Here we describe results that link copper homeostasis to capsule production and the inhibition of phagocytosis. Specifically, using Agrobacterium-mediated insertional mutagenesis, we identified an insertion in the promoter region of the putative copper transporter-encoding gene CTR2 that results in reduced expression of CTR2 and increased phagocytosis by murine RAW264.7 macrophages. The mutant also displayed sensitivity to copper starvation and defects in polysaccharide capsule production and melanization. These defects were all reversed by genetic correction of the promoter insertion by homologous targeting. Several melanization-defective mutants identified previously, those in the RIM20, RIM101, and VPS25 genes, also display sensitivity to copper starvation, reduced capsule production and increased phagocytosis. Together these results indicate a previously undescribed link between copper homeostasis to polysaccharide capsule production and phagocytosis inhibition in Cryptococcus neoformans

Ss-Sl2, a Novel Cell Wall Protein with PAN Modules, Is Essential for Sclerotial Development and Cellular Integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum