Enhanced Resistance to Fungal and Bacterial Diseases Due to Overexpression of BSR1, a Rice RLCK, in Sugarcane, Tomato, and Torenia.

Sugarcane smut caused by Sporisorium scitamineum is one of the most devastating sugarcane diseases. Furthermore, Rhizoctonia solani causes severe diseases in various crops including rice, tomato, potato, sugar beet, tobacco, and torenia. However, effective disease-resistant genes against these pathogens have not been identified in target crops. Therefore, the transgenic approach can be used since conventional cross-breeding is not applicable. Herein, the overexpression of BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice receptor-like cytoplasmic kinase, was conducted in sugarcane, tomato and torenia. BSR1-overexpressing tomatoes exhibited resistance to the bacteria Pseudomonas syringae pv. tomato DC3000 and the fungus R. solani, whereas BSR1-overexpressing torenia showed resistance to R. solani in the growth room. Additionally, BSR1 overexpression conferred resistance to sugarcane smut in the greenhouse. These three BSR1-overexpressing crops exhibited normal growth and morphologies except in the case of exceedingly high levels of overexpression. These results indicate that BSR1 overexpression is a simple and effective tool for conferring broad-spectrum disease resistance to many crops.

The CC-NB-LRR protein BSR1 from Brachypodium confers resistance to Barley stripe mosaic virus in gramineous plants by recognising TGB1 movement protein.

Although some nucleotide binding, leucine-rich repeat immune receptor (NLR) proteins conferring resistance to specific viruses have been identified in dicot plants, NLR proteins involved in viral resistance have not been described in monocots. We have used map-based cloning to isolate the CC-NB-LRR (CNL) Barley stripe mosaic virus (BSMV) resistance gene barley stripe resistance 1 (BSR1) from Brachypodium distachyon Bd3-1 inbred line. Stable BSR1 transgenic Brachypodium line Bd21-3, barley (Golden Promise) and wheat (Kenong 199) plants developed resistance against BSMV ND18 strain. Allelic variation analyses indicated that BSR1 is present in several Brachypodium accessions collected from countries in the Middle East. Protein domain swaps revealed that the intact LRR domain and the C-terminus of BSR1 are required for resistance. BSR1 interacts with the BSMV ND18 TGB1 protein in planta and shows temperature-sensitive antiviral resistance. The R390 and T392 residues of TGB1ND (ND18 strain) and the G196 and K197 residues within the BSR1 P-loop motif are key amino acids required for immune activation. BSR1 is the first cloned virus resistance gene encoding a typical CNL protein in monocots, highlighting the utility of the Brachypodium model for isolation and analysis of agronomically important genes for crop improvement.

Breeding and characterization of the world's first practical rice variety with resistance to brown spot (Bipolaris oryzae) bred using marker-assisted selection.

Brown spot (BS) caused by Bipolaris oryzae is a serious disease of rice and decreases grain yield. Breeding for BS resistance is an economical solution but has not hitherto been achieved. To develop a practical BS-resistant variety, we introduced a chromosomal segment including a quantitative trait locus (QTL) for BS resistance, qBSfR11, derived from the BS-resistant local resource 'Tadukan', into the genetic background of the high-yielding but susceptible 'Mienoyume'. Resistance is controlled by a single recessive gene in a 1.3-Mbp region. We named this gene bsr1 (brown spot resistance 1). The near-isogenic line bsr1-NIL had a greater yield with larger grain width than Mienoyume but similar other agronomic traits in fields where BS was mild; it had a significantly lower BS disease score and a 28.8% higher yield in fields where BS was more severe, and it showed resistance to multiple isolates of BS fungus. It showed stable resistance to BS and had excellent agricultural traits in the presence of BS. We developed the bsr1-NIL with resistance to BS and applied it for variety registration to Ministry of Agriculture, Forestry and Fisheries in Japan as 'Mienoyume BSL'. This is the first report for the BS resistant rice variety bred using marker-assisted selection.

Tyrosine phosphorylation of a receptor-like cytoplasmic kinase, BSR1, plays a crucial role in resistance to multiple pathogens in rice.

Plants have evolved many receptor-like cytoplasmic kinases (RLCKs) to modulate their growth, development, and innate immunity. Broad-Spectrum Resistance 1 (BSR1) encodes a rice RLCK, whose overexpression confers resistance to multiple diseases, including fungal rice blast and bacterial leaf blight. However, the mechanisms underlying resistance remain largely unknown. In the present study, we report that BSR1 is a functional protein kinase that autophosphorylates and transphosphorylates an artificial substrate in vitro. Although BSR1 is classified as a serine/threonine kinase, it was shown to autophosphorylate on tyrosine as well as on serine/threonine residues when expressed in bacteria, demonstrating that it is a dual-specificity kinase. Protein kinase activity was found to be indispensable for resistance to rice blast and leaf blight in BSR1-overexpressing plants. Importantly, tyrosine phosphorylation of BSR1 was critical for proper localization of BSR1 in rice cells and played a crucial role in BSR1-mediated resistance to multiple diseases, as evidenced by compromised disease resistance in transgenic plants overexpressing a mutant BSR1 in which Tyr-63 was substituted with Ala. Overall, our data indicate that BSR1 is a non-receptor dual-specificity kinase and that both tyrosine and serine/threonine kinase activities are critical for the normal functioning of BSR1 in the resistance to multiple pathogens. Our results support the notion that tyrosine phosphorylation plays a major regulatory role in the transduction of defense signals from cell-surface receptor complexes to downstream signaling components in plants.

Improvement of Broad-Spectrum Disease-Resistant Rice by the Overexpression of BSR1 via a Moderate-Strength Constitutive Promoter and a Pathogen-Inducible Promoter.

Conferring crops with resistance to multiple diseases is crucial for stable food production. Genetic engineering is an effective means of achieving this. The rice receptor-like cytoplasmic kinase BSR1 mediates microbe-associated molecular pattern-induced immunity. In our previous study, we demonstrated that rice lines overexpressing BSR1 under the control of the maize ubiquitin promoter exhibited broad-spectrum resistance to rice blast, brown spot, leaf blight, and bacterial seedling rot. However, unfavorable phenotypes were observed, such as a decreased seed germination rate and a partial darkening of husked rice. Herein, we present a strategy to address these unfavorable phenotypes using an OsUbi7 constitutive promoter with moderate expression levels and a pathogen-inducible PR1b promoter. Rice lines expressing BSR1 under the influence of both promoters maintained broad-spectrum disease resistance. The seed germination rate and coloration of husked rice were similar to those of the wild-type rice.

Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species have very similar morphological characteristics and are difficult or impossible to distinguish using light microscopy. DNA sequencing has been an important tool in the definition of Gambierdiscus species, but it can be time-consuming and relatively expensive. To provide an alternative approach, a PCR-RFLP protocol was developed for efficient, rapid, and cost-effective identification of Gambierdiscus strains isolated from the Gulf of Mexico and Caribbean Sea, where CFP cases and Gambierdiscus spp. have been reported. The assay targets the D1-D2 hypervariable regions of the large subunit ribosomal RNA gene and uses a single restriction enzyme, BsrI. This method produces distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea, and also distinguishes them from four Pacific endemic species. This method was successfully used to type 465 clonal isolates of Gambierdiscus from the U.S. Virgin Islands and Akumal Beach - Mexico This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.