Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal. We describe protocols to generate stable targeted mutations by allelic exchange in Ehrlichia chaffeensis, an obligate intracellular tick-borne bacterium responsible for human monocytic ehrlichiosis. Targeted mutations in E. chaffeensis were created to disrupt two genes, and also to restore one gene by another allelic exchange mutation leading to the restoration of transcription and protein expression from the inactivated gene and the recovered organisms also express mCherry, which distinguishes from the wild type. We expect that the methods developed are broadly applicable to other obligates, particularly to rickettsial pathogens, to routinely perform targeted mutations to enable studies focused on protein structure-function analyses, host-pathogen interactions and in developing vaccines.

Abscisic acid (ABA) is an essential phytohormone involved in diverse physiological processes. Although genome-wide analyses of the ABA receptor PYR/PYL/RCAR (PYL) protein/gene family have been performed in certain plant species, little is known about the ABA receptor protein/gene family in the rubber tree (Hevea brasiliensis). In this study, we identified 14 ABA receptor PYL proteins/genes (designated HbPYL1 through HbPYL14) in the most recent rubber tree genome. A phylogenetic tree was constructed, which demonstrated that HbPYLs can be divided into three subfamilies that correlate well with the corresponding Arabidopsis subfamilies. Eight HbPYLs are highly expressed in laticifers. Five of the eight genes are simultaneously regulated by ABA, jasmonic acid (JA) and ethylene (ET). The identification and characterization of HbPYLs should enable us to further understand the role of ABA signal in the rubber tree.

Newcastle disease (ND) has a great impact on poultry health and welfare with its most virulent (velogenic) strain. In addition, issues exacerbated by the increase in global temperatures necessitates a greater understanding of the host immune response when facing a combination of biotic and abiotic stress factors in poultry production. Previous investigations have revealed that the host immune response is tissue-specific. The goal of this study was to identify genes and/or signaling pathways associated with immune response to NDV (Newcastle disease virus) in the trachea, an essential organ where NDV replicate after the infection, by profiling the tissue specific transcriptome response in two genetically distinct inbred chicken lines when exposed to both abiotic and biotic stressors. Fayoumis appear to be able to respond more effectively (lower viral titer, higher antibody levels, immune gene up-regulation) and earlier than Leghorns. Our results suggest NDV infection in Fayoumis appears to elicit proinflammatory processes, and pathways such as the inhibition of cell viability, cell proliferation of lymphocytes, and transactivation of RNA, more rapidly than in Leghorns. These differences in immune response converge at later timepoints which may indicate that Leghorns eventually regulate its immune response to infection. The profiling of the gene expression response in the trachea adds to our understanding of the chicken host response to NDV infection and heat stress on a whole genome level and provides potential candidate genes and signaling pathways for further investigation into the characterization of the time-specific and pathway specific responses in Fayoumis and Leghorns.

N-myristoyltransferase-1 (NMT1) catalyzes protein myristoylation, a lipid modification that is elevated in cancer cells. NMT1 sustains proliferation and/or survival of cancer cells through mechanisms that are not completely understood. We used genetic and pharmacological inhibition of NMT1 to further dissect the role of this enzyme in cancer, and found an unexpected essential role for NMT1 at promoting lysosomal metabolic functions. Lysosomes mediate enzymatic degradation of vesicle cargo, and also serve as functional platforms for mTORC1 activation. We show that NMT1 is required for both lysosomal functions in cancer cells. Inhibition of NMT1 impaired lysosomal degradation leading to autophagy flux blockade, and simultaneously caused the dissociation of mTOR from the surface of lysosomes leading to decreased mTORC1 activation. The regulation of lysosomal metabolic functions by NMT1 was largely mediated through the lysosomal adaptor LAMTOR1. Accordingly, genetic targeting of LAMTOR1 recapitulated most of the lysosomal defects of targeting NMT1, including defective lysosomal degradation. Pharmacological inhibition of NMT1 reduced tumor growth, and tumors from treated animals had increased apoptosis and displayed markers of lysosomal dysfunction. Our findings suggest that compounds targeting NMT1 may have therapeutic benefit in cancer by preventing mTORC1 activation and simultaneously blocking lysosomal degradation, leading to cancer cell death.