Understanding how the Effectors HopD1 and HopG1 from the Bacterial Pathogen Pseudomonas syringae pv. tomato DC3000 target the Arabidopsis thaliana Protein AtNHR2B to cause Disease in Plants

The pathogenicity of Pseudomonas syringae is associated with the type III secretion system (T3SS), a complex of proteins assembled in the inner and outer bacterial membranes that traverses the plant cell wall to deliver bacterial proteins into the cytoplasm of plant cells. The effector proteins translocated into the plant cells are called Hops (Hypersensitive response and pathogenicity outer proteins). Bacterial effectors target plant immune proteins to suppress defense responses and enhance bacterial parasitism. The Arabidopsis thaliana nonhost resistance 2B (AtNHR2B), a recently identified immune protein, is degraded after inoculation with the adapted pathogen of Arabidopsis, P. syringae pv tomato DC3000 (Pst DC3000), but not by the non-adapted pathogen P. syringae pv. tabaci (Pstab). Several Pst DC3000 effectors, including HopG1 and HopD1 interact with AtNHR2B in planta. Characterization of the effectors presence in plants upon inoculation with Pstab showed that transgenic expression of HopG1-FLAG triggered cell death, high electrolyte leakage levels and increased production of mitochondrial ROS. In contrast, HopG1-FLAG expression in combination with AtNHR2B-GFP caused susceptibility to Pstab as shown by the development of disease symptoms and the significant increase in bacterial growth. Together, these results suggest that HopG1 targets AtNHR2B to interfere with plant immune response upon bacterial infection. In contrast, transgenic plants expressing HopD1-HA alone or in combination with AtNHR2B-GFP, showed disease symptoms after inoculation with Pstab, that normally does not cause disease in wild-type Col-0 plants. Moreover, Pstab grew significantly more in transgenic plants overexpressing HopD1-HA than in wild-type Col-0. Interestingly, Arabidopsis plants expressing the bacterial effector HopD1-HA alone or in combination with AtNHR2B-GFP were deficient in callose deposition and showed a downregulation of the callose synthase gene PMR4. Altogether, these results suggest that HopD1 interferes with callose deposition and by doing so hinders defense responses

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field