Marker-Assisted Introgression of the Salinity Tolerance Locus Saltol in Temperate Japonica Rice.

BACKGROUND: Rice is one of the most salt sensitive crops at seedling, early vegetative and reproductive stages. Varieties with salinity tolerance at seedling stage promote an efficient growth at early stages in salt affected soils, leading to healthy vegetative growth that protects crop yield. Saltol major QTL confers capacity to young rice plants growing under salt condition by maintaining a low Na+/K+ molar ratio in the shoots. RESULTS: Marker-assisted backcross (MABC) procedure was adopted to transfer Saltol locus conferring salt tolerance at seedling stage from donor indica IR64-Saltol to two temperate japonica varieties, Vialone Nano and Onice. Forward and background selections were accomplished using polymorphic KASP markers and a final evaluation of genetic background recovery of the selected lines was conducted using 15,580 SNP markers obtained from Genotyping by Sequencing. Three MABC generations followed by two selfing, allowed the identification of introgression lines achieving a recovery of the recurrent parent (RP) genome up to 100% (based on KASP markers) or 98.97% (based on GBS). Lines with highest RP genome recovery (RPGR) were evaluated for agronomical-phenological traits in field under non-salinized conditions. VN1, VN4, O1 lines were selected considering the agronomic evaluations and the RPGR% results as the most interesting for commercial exploitation. A physiological characterization was conducted by evaluating salt tolerance under hydroponic conditions. The selected lines showed lower standard evaluation system (SES) scores: 62% of VN4, and 57% of O1 plants reaching SES 3 or SES 5 respectively, while only 40% of Vialone Nano and 25% of Onice plants recorded scores from 3 to 5, respectively. VN1, VN4 and O1 showed a reduced electrolyte leakage values, and limited negative effects on relative water content and shoot/root fresh weight ratio. CONCLUSION: The Saltol locus was successfully transferred to two elite varieties by MABC in a time frame of three years. The application of background selection until BC3F3 allowed the selection of lines with a RPGR up to 98.97%. Physiological evaluations for the selected lines indicate an improved salinity tolerance at seedling stage. The results supported the effectiveness of the Saltol locus in temperate japonica and of the MABC procedure for recovering of the RP favorable traits.

Phosphate accumulation in rice leaves promotes fungal pathogenicity and represses host immune responses during pathogen infection.

Rice is one of the most important crops in the world and a staple food for more than half of the world's population. At present, the blast disease caused by the fungus Magnaporthe oryzae poses a severe threat to food security through reduction of rice yields worldwide. High phosphate fertilization has previously been shown to increase blast susceptibility. At present, however, our knowledge on the mechanisms underpinning phosphate-induced susceptibility to M. oryzae infection in rice is limited. In this work, we conducted live cell imaging on rice sheaths inoculated with a M. oryzae strain expressing two fluorescently-tagged M. oryzae effectors. We show that growing rice under high phosphate fertilization, and subsequent accumulation of phosphate in leaf sheaths, promotes invasive growth of M. oryzae. Consistent with this, stronger expression of M. oryzae effectors and Pathogenicity Mitogen-activated Protein Kinase (PMK1) occurs in leaf sheaths of rice plants grown under high a phosphate regime. Down-regulation of fungal genes encoding suppressors of plant cell death and up-regulation of plant cell death-inducing effectors also occurs in sheaths of phosphate over-accumulating rice plants. Treatment with high Pi causes alterations in the expression of fungal phosphate transporter genes potentially contributing to pathogen virulence. From the perspective of the plant, Pi accumulation in leaf sheaths prevents H2O2 accumulation early during M. oryzae infection which was associated to a weaker activation of Respiratory Burst Oxidase Homologs (RBOHs) genes involved in reactive oxygen species (ROS) production. Further, a weaker activation of defense-related genes occurs during infection in rice plants over-accumulating phosphate. From these results, it can be concluded that phosphate fertilization has an effect on the two interacting partners, pathogen and host. Phosphate-mediated stimulation of fungal effector genes (e.g., potentiation of fungal pathogenicity) in combination with repression of pathogen-inducible immune responses (e.g., ROS accumulation, defense gene expression) explains higher colonization by M. oryzae in rice tissues accumulating phosphate. Phosphate content can therefore be considered as an important factor in determining the outcome of the rice/M. oryzae interaction. As fertilizers and pesticides are commonly used in rice cultivation to maintain optimal yield and to prevent losses caused by pathogens, a better understanding of how phosphate impacts blast susceptibility is crucial for developing strategies to rationally optimize fertilizer and pesticide use in rice production.

Loss-of-function of NITROGEN LIMITATION ADAPTATION confers disease resistance in Arabidopsis by modulating hormone signaling and camalexin content.

Phosphorus is an important macronutrient required for plant growth and development. It is absorbed by the roots in the form of inorganic phosphate (Pi). Under Pi limiting conditions, plants activate the Phosphate Starvation Response (PSR) system to enhance Pi acquisition. The NITROGEN LIMITATION ADAPTION (NLA) gene is a component of the Arabidopsis PSR, and its expression is post-transcriptionally regulated by miR827. We show that loss-of-function of NLA and MIR827 overexpression increases Pi level and enhances resistance to infection by the fungal pathogen Plectosphaerella cucumerina in Arabidopsis. Upon pathogen infection, high Pi plants (e.g. nla plants and wild type plants grown under high Pi supply) showed enhanced callose deposition. High Pi plants also exhibited superinduction of camalexin biosynthesis genes which is consistent with increased levels of camalexin during pathogen infection. Pathogen infection and treatment with fungal elicitors, triggered up-regulation of MIR827 and down-regulation of NLA expression. Under non-infection conditions, the nla plants showed increased levels of SA and JA compared with wild type plants, their levels further increasing upon pathogen infection. Overall, the outcomes of this study suggest that NLA plays a role in Arabidopsis immunity, while supporting convergence between Pi signaling and immune signaling in Arabidopsis.

Phosphate-induced resistance to pathogen infection in Arabidopsis.

In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss-of-function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling pathways were higher in high Pi plants compared to wild-type plants grown under control conditions, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses.

Integrative Approach for Precise Genotyping and Transcriptomics of Salt Tolerant Introgression Rice Lines.

Rice is the most salt sensitive cereal crop and its cultivation is particularly threatened by salt stress, which is currently worsened due to climate change. This study reports the development of salt tolerant introgression lines (ILs) derived from crosses between the salt tolerant indica rice variety FL478, which harbors the Saltol quantitative trait loci (QTL), and the salt-sensitive japonica elite cultivar OLESA. Genotyping-by-sequencing (GBS) and Kompetitive allele specific PCR (KASPar) genotyping, in combination with step-wise phenotypic selection in hydroponic culture, were used for the identification of salt-tolerant ILs. Transcriptome-based genotyping allowed the fine mapping of indica genetic introgressions in the best performing IL (IL22). A total of 1,595 genes were identified in indica regions of IL22, which mainly located in large introgressions at Chromosomes 1 and 3. In addition to OsHKT1;5, an important number of genes were identified in the introgressed indica segments of IL22 whose expression was confirmed [e.g., genes involved in ion transport, callose synthesis, transcriptional regulation of gene expression, hormone signaling and reactive oxygen species (ROS) accumulation]. These genes might well contribute to salt stress tolerance in IL22 plants. Furthermore, comparative transcript profiling revealed that indica introgressions caused important alterations in the background gene expression of IL22 plants (japonica cultivar) compared with its salt-sensitive parent, both under non-stress and salt-stress conditions. In response to salt treatment, only 8.6% of the salt-responsive genes were found to be commonly up- or down-regulated in IL22 and OLESA plants, supporting massive transcriptional reprogramming of gene expression caused by indica introgressions into the recipient genome. Interactions among indica and japonica genes might provide novel regulatory networks contributing to salt stress tolerance in introgression rice lines. Collectively, this study illustrates the usefulness of transcriptomics in the characterization of new rice lines obtained in breeding programs in rice.

Phosphate excess increases susceptibility to pathogen infection in rice.

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease.

Rice Seeds as Biofactories of Rationally Designed and Cell-Penetrating Antifungal PAF Peptides.

PAFs are short cationic and tryptophan-rich synthetic peptides with cell-penetrating antifungal activity. They show potent and selective killing activity against major fungal pathogens and low toxicity to other eukaryotic and bacterial cells. These properties make them a promising alternative to fulfill the need of novel antifungals with potential applications in crop protection, food preservation, and medical therapies. However, the difficulties of cost-effective manufacturing of PAFs by chemical synthesis or biotechnological production in microorganisms have hampered their development for practical use. This work explores the feasibility of using rice seeds as an economical and safe production system of PAFs. The rationally designed PAF102 peptide with improved antifungal properties was selected for assessing PAF biotechnological production. Two different strategies are evaluated: (1) the production as a single peptide targeted to protein bodies and (2) the production as an oleosin fusion protein targeted to oil bodies. Both strategies are designed to offer stability to the PAF peptide in the host plant and to facilitate its downstream purification. Our results demonstrate that PAF does not accumulate to detectable levels in rice seeds when produced as a single peptide, whereas it is successfully produced as fusion protein to the Oleosin18, up to 20 µg of peptide per gram of grain. We show that the expression of the chimeric Ole18-PAF102 gene driven by the Ole18 promoter results in the specific accumulation of the fusion protein in the embryo and aleurone layer of the rice seed. Ole18-PAF102 accumulation has no deleterious effects on seed yield, germination capacity, or seedling growth. We also show that the Oleosin18 protein serves as carrier to target the fusion protein to oil bodies facilitating PAF102 recovery. Importantly, the recovered PAF102 is active against the fungal phytopathogen Fusarium proliferatum. Altogether, our results prove that the oleosin fusion technology allows the production of PAF bioactive peptides to assist the exploitation of these antifungal compounds.

A Critical Role of Sodium Flux via the Plasma Membrane Na+/H+ Exchanger SOS1 in the Salt Tolerance of Rice.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na+) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na+/H+ exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1 In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress.

Calcium-dependent protein kinase OsCPK10 mediates both drought tolerance and blast disease resistance in rice plants.

Plant growth and productivity is negatively affected by different stresses. Most stresses trigger calcium signals that initiate acclimation responses in plants. The multigene family of plant calcium-dependent protein kinases (CPKs) functions in multiple stress responses by transducing calcium signals into phosphorylation events. This work reports that the OsCPK10 isoform positively mediates tolerance to different stresses in rice plants by enhancing their antioxidant capacity and protecting them from reactive oxygen species (ROS) damage, with the uncontrolled generation of ROS being a common feature of these stresses. Here, we show that the constitutive accumulation of an HA-tagged OsCPK10 full-length protein enhances the hydrogen peroxide detoxifying capacity of rice plants during desiccation. This is achived by modulating the accumulation of catalase proteins, which reduces the extent of lipid peroxidation and protects the integrity of cell membranes, resulting in drought tolerance. OsCPK10HA accumulation also confers blast disease resistance by interfering with fungal necrotrophic growth via a reduction in the accumulation of hydrogen peroxide. Furthermore, we show by bimolecular complementation assays that OsCPK10 is a plasma membrane protein that physically interacts in vivo with catalase A. OsCPK10 therefore appears to be a good molecular target to improve tolerance to abiotic stresses as well as to blast disease, which limit rice crop productivity.

Production of BP178, a derivative of the synthetic antibacterial peptide BP100, in the rice seed endosperm.

BACKGROUND: BP178 peptide is a synthetic BP100-magainin derivative possessing strong inhibitory activity against plant pathogenic bacteria, offering a great potential for future applications in plant protection and other fields. Here we report the production and recovery of a bioactive BP178 peptide using rice seeds as biofactories. RESULTS: A synthetic gene encoding the BP178 peptide was prepared and introduced in rice plants. The gene was efficiently expressed in transgenic rice under the control of an endosperm-specific promoter. Among the three endosperm-specific rice promoters (Glutelin B1, Glutelin B4 or Globulin 1), best results were obtained when using the Globulin 1 promoter. The BP178 peptide accumulated in the seed endosperm and was easily recovered from rice seeds using a simple procedure with a yield of 21 µg/g. The transgene was stably inherited for at least three generations, and peptide accumulation remained stable during long term storage of transgenic seeds. The purified peptide showed in vitro activity against the bacterial plant pathogen Dickeya sp., the causal agent of the dark brown sheath rot of rice. Seedlings of transgenic events showed enhanced resistance to the fungal pathogen Fusarium verticillioides, supporting that the in planta produced peptide was biologically active. CONCLUSIONS: The strategy developed in this work for the sustainable production of BP178 peptide using rice seeds as biofactories represents a promising system for future production of peptides for plant protection and possibly in other fields.

Production of Biologically Active Cecropin A Peptide in Rice Seed Oil Bodies.

Cecropin A is a natural antimicrobial peptide that exhibits fast and potent activity against a broad spectrum of pathogens and neoplastic cells, and that has important biotechnological applications. However, cecropin A exploitation, as for other antimicrobial peptides, is limited by their production and purification costs. Here, we report the efficient production of this bioactive peptide in rice bran using the rice oleosin 18 as a carrier protein. High cecropin A levels were reached in rice seeds driving the expression of the chimeric gene by the strong embryo-specific oleosin 18 own promoter, and targeting the peptide to the oil body organelle as an oleosin 18-cecropin A fusion protein. The accumulation of cecropin A in oil bodies had no deleterious effects on seed viability and seedling growth, as well as on seed yield. We also show that biologically active cecropin A can be easily purified from the transgenic rice seeds by homogenization and simple flotation centrifugation methods. Our results demonstrate that the oleosin fusion technology is suitable for the production of cecropin A in rice seeds, which can potentially be extended to other antimicrobial peptides to assist their exploitation.

Enhancing blast disease resistance by overexpression of the calcium-dependent protein kinase OsCPK4 in rice.

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium-dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.

Production of cecropin A antimicrobial peptide in rice seed endosperm.

BACKGROUND: Cecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds. RESULTS: Transgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active. CONCLUSIONS: Rice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.