Investigating the biology of rice blast disease and prospects for durable resistance

There are important biological process involved in rice blast disease that are now well-studied during the early events in plant infection which include: the cell biology of appressorium formation, the biology of invasive growth and effector secretion, the two distinct mechanisms of effector secretion, the nature of the plant-pathogen interface, PAMP-triggered immunity modulation by secreted effectors and effector-triggered immunity and blast resistance. The devastating losses caused by the blast fungus have been documented in most grasses, but this chapter discusses the use of major resistance genes to rice blast and wheat blast disease as an emerging threat to global food security. This chapter also highlights an emerging approach to breed for durable resistance to plant pathogens using gene editing technologies with an example: CRISPR-Cas9 mutagenesis of dominant S-genes for disease control.

CesL Regulates Type III Secretion Substrate Specificity of the Enteropathogenic E. coli Injectisome

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Salmonella enters a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence.

The Type III Accessory Protein HrpE of Xanthomonas oryzae pv. oryzae Surpasses the Secretion Role, and Enhances Plant Resistance and Photosynthesis

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution

Many bacteria employ a type III secretion system (T3SS), also called injectisome, to translocate proteins into eukaryotic host cells through a hollow extracellular needle. The system can efficiently transport heterologous cargo, which makes it a uniquely suited tool for the translocation of proteins into eukaryotic cells. However, the injectisome indiscriminately injects proteins into any adjoining eukaryotic cell, and this lack of target specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to control protein secretion and translocation into eukaryotic cells by light. By combining optogenetic interaction switches with the dynamic cytosolic T3SS component SctQ, the cytosolic availability of SctQ and in consequence T3SS-dependent effector secretion can be regulated by external light. The resulting system, which we call LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the application of the system for light-regulated translocation of a heterologous reporter protein into cultured eukaryotic cells. LITESEC-T3SS represents a new method to achieve unparalleled spatial and temporal resolution for the controlled protein translocation into eukaryotic host cells.