Differential Expression of Fungal Genes Determines the Lifestyle of Plectosphaerella Strains During Arabidopsis thaliana Colonization.

The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.

CRISPR/Cas9-mediated deletion of the Wiskott-Aldrich syndrome locus causes actin cytoskeleton disorganization in murine erythroleukemia cells.

Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing the Was gene in the X chromosome of murine erythroleukemia (MEL) cells. We show that Was-deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoring Was expression. We found that whereas the total amount of actin protein was similar between wild-type and Was knockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate that Was overexpression can mediate the activation of Bruton's tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.

Analysis of DNA topology of EBV minichromosomes in HEK 293 cells.

Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3'Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV "family of repeats" induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner.

Differential gene expression analysis by RNA-seq reveals the importance of actin cytoskeletal proteins in erythroleukemia cells.

Development of drug resistance limits the effectiveness of anticancer treatments. Understanding the molecular mechanisms triggering this event in tumor cells may lead to improved therapeutic strategies. Here we used RNA-seq to compare the transcriptomes of a murine erythroleukemia cell line (MEL) and a derived cell line with induced resistance to differentiation (MEL-R). RNA-seq analysis identified a total of 596 genes (Benjamini-Hochberg adjusted p-value < 0.05) that were differentially expressed by more than two-fold, of which 81.5% (486/596) of genes were up-regulated in MEL cells and 110 up-regulated in MEL-R cells. These observations revealed that for some genes the relative expression of mRNA amount in the MEL cell line has decreased as the cells acquired the resistant phenotype. Clustering analysis of a group of genes showing the highest differential expression allowed identification of a sub-group among genes up-regulated in MEL cells. These genes are related to the organization of the actin cytoskeleton network. Moreover, the majority of these genes are preferentially expressed in the hematopoietic lineage and at least three of them, Was (Wiskott Aldrich syndrome), Btk (Bruton's tyrosine kinase) and Rac2, when mutated in humans, give rise to severe hematopoietic deficiencies. Among the group of genes that were up-regulated in MEL-R cells, 16% of genes code for histone proteins, both canonical and variants. A potential implication of these results on the blockade of differentiation in resistant cells is discussed.