Spontaneously Blinking Fluorescent Protein for Simple Single Laser Super-Resolution Live Cell Imaging.

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

Genetic organization of the hrp gene cluster in Acidovorax avenae strain N1141 and a novel effector protein that elicits immune responses in rice (Oryza sativa L.).

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.

Rice cDNA microarray-based gene expression profiling of the response to flagellin perception in cultured rice cells.

Incompatible strains of Acidovorax avenae elicit an immune response in cultured rice cells, with immunity specifically induced by the flagellin of the incompatible strain. To identify genes regulated by flagellin perception signaling in cultured rice cells, gene expression patterns were analyzed with rice cDNA microarrays, including 3,353 independent rice cDNA clones. In all, 131 genes were differentially expressed between incompatible and compatible interactions. K-means clustering showed that 94 genes were upregulated and 32 genes were downregulated during incompatible interactions, whereas only 5 genes were upregulated during compatible interactions. Among the 126 genes that were up- or downregulated during incompatible interactions, expression of 46 genes was decreased when cultured rice cells were inoculated with a flagellin-deficient incompatible strain (delta fla1141-2), indicating that approximately 37% of the 126 genes were directly controlled by flagellin perception. Real-time reverse-transcription polymerase chain reaction analysis using flagellins purified from incompatible or compatible strains was performed to confirm flagellin-regulated expression of candidate genes selected by microarray analysis. Results showed that induction of some genes involved in the immune response is regulated not only by the flagellin perception pathway, but also by another recognition molecule-perception pathway.

Flagellin from an incompatible strain of Acidovorax avenae mediates H2O2 generation accompanying hypersensitive cell death and expression of PAL, Cht-1, and PBZ1, but not of Lox in rice.

Acidovorax avenae causes a brown stripe disease in monocot plants. We recently reported that a rice-incompatible strain of A. avenae caused hypersensitive cell death in rice and that the flagellin of the incompatible strain was involved in this response. The incompatible strain induced the rapid generation of H2O2 accompanying hypersensitive cell death and the expression of defense genes such as PAL, Cht-1, PBZ1, and LOX, whereas the compatible strain did not. The purified incompatible flagellin also induced the expression of PAL, Cht-1, and PBZ1, but LOX expression was not induced by the incompatible flagellin. PAL and LOX enzymatic activities were increased by inoculation with the incompatible strain, whereas only PAL activity was increased by the incompatible flagellin. Interestingly, the flagellin-deficient incompatible strain lost the ability to generate H2O2 and induce hypersensitive cell death, but PAL, Cht-1, and PBZ1 expression still were induced by inoculation with the deficient strain, suggesting that induction of these genes is regulated not only by flagellin but also by some other signal. Thus, the incompatible flagellin of A. avenae is a specific elicitor in rice, but it is not the only factor capable of inducing the rice defense system.