Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.
Agrobacterium tumefaciens/metabolism* ; Alleles ; Arabidopsis/metabolism* ; Arabidopsis Proteins/genetics* ; Gene Expression Regulation, Plant ; Genetic Techniques ; Humans ; Lipopolysaccharides/metabolism* ; Luminescence ; Mutation ; NADPH Oxidase 2/chemistry* ; NADPH Oxidases/genetics* ; Plant Stomata/metabolism* ; Protein Domains ; Reactive Oxygen Species/metabolism* ; Nicotiana/metabolism*

Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China.
Animals ; Antibodies, Monoclonal/immunology* ; China ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay/methods* ; Female ; Hybridomas ; Solanum lycopersicum/virology* ; Mice ; Mice, Inbred BALB C ; Plant Diseases/virology* ; Potexvirus/metabolism* ; Sensitivity and Specificity ; Nicotiana