Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H₂O₂ accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity.

The plant NADPH oxidases produce reactive oxygen species (ROS) in response to pathogens that have diverse functions in different cellular contexts. Distinct phenotypic outcomes may derive from the interaction of NADPH oxidase-dependent ROS with other signaling components that mediate defense activation. We analyze the interaction between NADPH oxidases AtRbohD and AtRbohF and the Arabidopsis heterotrimeric G protein. The Gβ subunit (AGB1) of the heterotrimeric G protein is required for full disease resistance to different Pseudomonas syringae strains. Genetic studies reveal that, upon P. syringae infection, AGB1 and AtRbohD and AtRbohF can function in the same pathway, as the agb1 null allele is epistatic to the NADPH oxidase null alleles, combinatorial mutants display the agb1 phenotypes, and agb1 suppresses some of the atrbohD atrbohF double mutant phenotypes. In contrast, increased susceptibility to the necrotrophic fungus Plectosphaerella cucumerina displayed by agb1 and atrbohD atrbohF is enhanced in the agb1 atrbohD atrbohF triple mutant, suggesting that NADPH oxidase and heterotrimeric G proteins mediate different response pathways in response to this necrotrophic pathogen. The defense response mediated by AGB1 is independent of pathogen-dependent salicylic acid accumulation and signaling, as the agb1 sid2 (isochorismate synthase 2) double mutant showed enhanced disease susceptibility to P. syringae and Plectosphaerella cucumerina as compared with both single mutants. This study exemplifies the complex interplay between signaling events mediating defense activation, depending on the type of plant-pathogen interaction.

Genetic ablation of the β subunit of the heterotrimeric G protein complex in agb1-2 confers defective activation of microbe-associated molecular pattern (MAMP)-triggered immunity, resulting in agb1-2 enhanced susceptibility to pathogens like the fungus Plectosphaerella cucumerina BMM. A mutant screen for suppressors of agb1-2 susceptibility (sgb) to P. cucumerina BMM identified sgb10, a new null allele (mkp1-2) of the mitogen-activated protein kinase phosphatase 1 (MKP1). The enhanced susceptibility of agb1-2 to the bacterium Pseudomonas syringae pv. tomato DC3000 and the oomycete Hyaloperonospora arabidopsidis is also abrogated by mkp1-2. MKP1 negatively balances production of reactive oxygen species (ROS) triggered by MAMPs, since ROS levels are enhanced in mkp1. The expression of RBOHD, encoding a NADPH oxidase–producing ROS, is upregulated in mkp1 upon MAMP treatment or pathogen infection. Moreover, MKP1 negatively regulates RBOHD activity, because ROS levels upon MAMP treatment are increased in mkp1 plants constitutively overexpressing RBOHD (35S::RBOHD mkp1). A significant reprograming of mkp1 metabolic profile occurs with more than 170 metabolites, including antimicrobial compounds, showing differential accumulation in comparison with wild-type plants. These results suggest that MKP1 functions downstream of the heterotrimeric G protein during MAMP-triggered immunity, directly regulating the activity of RBOHD and ROS production as well as other immune responses.

Bluetongue is an infectious, non-contagious, arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV), producing severe impacts on livestock. Historically, Southern Europe has suffered multiple incursions of different BTV serotypes with serious consequences. In 2021, BTV re-emerged in the Balearic Islands (Spain) after 16 years free of the disease, causing a large outbreak that mainly affected sheep, as well as cattle and goats. A novel emerging strain of BTV serotype 4 (BTV-4) was identified via preliminary molecular characterization as the etiological culprit of the epizootic. This study delineates the outbreak in the Balearic Islands in 2021, encompassing field-based clinical observations and laboratory findings. Additionally, an experimental infection was conducted in sheep using the novel BTV-4 strain to assess its virulence, pathogenicity, and laboratory diagnostic characteristics. The in vivo characterization was conducted concurrently with the virulent and widely disseminated BTV-4 RNM 2020 strain that has precipitated significant outbreaks in the Mediterranean region in recent years. Both strains exhibited analogous pathogenic potential in sheep and yielded equivalent outcomes in diagnostic parameters. Furthermore, the impact of the novel BTV-4 strain is discussed.

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease of ruminants, and has a severe impact on livestock. It is caused by Bluetongue virus (BTV), a double-stranded (ds) RNA virus with a segmented genome (10 segments), belonging to the Seoreoviridae family, Orbivirus genus. Over the last 25 years, Europe has suffered multiple incursions of different BTV serotypes with serious consequences, which have mainly been controlled thanks to vaccination. In the case of Spain, from 2000 to 2023, BTV serotypes 1, 2, 4 and 8 have caused epidemics, and, sporadically, BTV-1 and -4 were detected in the same area and period. In 2024, BTV serotypes 1, 3 and 8 circulated simultaneously in the southwest of the country, causing a severe clinical impact in sheep but also in cattle and a multitude of outbreaks. Additionally, despite vaccination, serotype 4 also circulated that year, especially in areas where the other serotypes were already circulating. Whole-genome sequencing and phylogenetic analyses allowed us to confirm that serotypes 1 and 4 were homologous to viruses circulating in the country since 2000s, while serotypes 3 and 8 were homologous to BTVs recently notified in neighboring countries. In this context, many BTV co-infections of two or more different serotypes were confirmed by serotype-specific RT-PCRs, both in farms and individual animals. An epidemic caused by four serotypes coinciding in space and time had never occurred before in Spain, being a challenge for the diagnosis and control of this disease. Moreover, it could have favored the appearance of reassortant viruses with an unknown virulence, posing an additional risk. The data presented here raise the question of whether the co-circulation of different BTV strains, an exceptional situation, could become the new normal in certain areas of Europe.