A novel Transposable element-derived microRNA participates in plant immunity to rice blast disease.

MicroRNAs (miRNAs) are small non-coding RNAs that direct post-transcriptional gene silencing in plant development and stress responses through cleavage or translational repression of target mRNAs. Here, we report the identification and functional characterization of a new member of the miR812 family in rice (named as miR812w) involved in disease resistance. miR812w is present in cultivated Oryza species, both japonica and indica subspecies, and wild rice species within the Oryza genus, but not in dicotyledonous species. miR812w is a 24nt-long that requires DCL3 for its biogenesis and is loaded into AGO4 proteins. Whereas overexpression of miR812w increased resistance to infection by the rice blast fungus Magnaporthe oryzae, CRISPR/Cas9-mediated MIR812w editing enhances disease susceptibility, supporting that miR812w plays a role in blast resistance. We show that miR812w derives from the Stowaway type of rice MITEs (Miniature Inverted-Repeat Transposable Elements). Moreover, miR812w directs DNA methylation in trans at target genes that have integrated a Stowaway MITE copy into their 3' or 5' untranslated region (ACO3, CIPK10, LRR genes), as well as in cis at the MIR812w locus. The target genes of miR812 were found to be hypo-methylated around the miR812 recognition site, their expression being up-regulated in transgene-free CRISPR/Cas9-edited miR812 plants. These findings further support that, in addition to post-transcriptional regulation of gene expression, miRNAs can exert their regulatory function at the transcriptional level. This relationship between miR812w and Stowaway MITEs integrated into multiple coding genes might eventually create a network for miR812w-mediated regulation of gene expression with implications in rice immunity.

Osa-miR7695 enhances transcriptional priming in defense responses against the rice blast fungus.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level in eukaryotes. In rice, MIR7695 expression is regulated by infection with the rice blast fungus Magnaporthe oryzae with subsequent down-regulation of an alternatively spliced transcript of natural resistance-associated macrophage protein 6 (OsNramp6). NRAMP6 functions as an iron transporter in rice. RESULTS: Rice plants grown under high iron supply showed blast resistance, which supports that iron is a factor in controlling blast resistance. During pathogen infection, iron accumulated in the vicinity of M. oryzae appressoria, the sites of pathogen entry, and in cells surrounding infected regions of the rice leaf. Activation-tagged MIR7695 rice plants (MIR7695-Ac) exhibited enhanced iron accumulation and resistance to M. oryzae infection. RNA-seq analysis revealed that blast resistance in MIR7695-Ac plants was associated with strong induction of defense-related genes, including pathogenesis-related and diterpenoid biosynthetic genes. Levels of phytoalexins during pathogen infection were higher in MIR7695-Ac than wild-type plants. Early phytoalexin biosynthetic genes, OsCPS2 and OsCPS4, were also highly upregulated in wild-type rice plants grown under high iron supply. CONCLUSIONS: Our data support a positive role of miR7695 in regulating rice immunity that further underpin links between defense and iron signaling in rice. These findings provides a basis to better understand regulatory mechanisms involved in rice immunity in which miR7695 participates which has a great potential for the development of strategies to improve blast resistance in rice.

OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice.

Background and Aims: MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem-loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated. Methods: Activation-tagged Osdcl1a (Osdcl1a-Ac) rice mutants were examined for resistance to pathogen infection. mRNA and small RNA deep sequencing, quantitative real-time PCR (RT-qPCR) and stem-loop reverse tanscripion-PCR (RT-PCR) were used to examine DCL1a-mediated alterations in the rice transcriptome. Rice diterpene phytoalexins were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Accumulation of O2·- was determined by nitroblue tetrazolium (NBT) staining. Key Results: dcl1a-Ac mutants exhibit enhanced susceptibility to infection by fungal pathogens which was associated with a weaker induction of defence gene expression. Comparison of the mRNA and miRNA transcriptomes of dcl1a-Ac and wild-type plants revealed misregulation of genes involved in detoxification of reactive oxygen species. Consequently, dcl1a-Ac plants accumulated O2·- in their leaves and were more sensitive to methyl viologen-induced oxidative stress. Furthermore, dcl1a-Ac plants showed downregulation of diterpenoid phytoalexin biosynthetic genes, these genes also being weakly induced during pathogen infection. Upon pathogen challenge, dcl1a-Ac plants failed to accumulate major diterpenoid phytoalexins. OsDCL1a activation resulted in marked alterations in the rice miRNAome, including both upregulation and downregulation of miRNAs. Conclusions: OsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity.

The MicroRNA miR773 Is Involved in the Arabidopsis Immune Response to Fungal Pathogens.

MicroRNAs (miRNAs) are 21- to 24-nucleotide short noncoding RNAs that trigger gene silencing in eukaryotes. In plants, miRNAs play a crucial role in a wide range of developmental processes and adaptive responses to abiotic and biotic stresses. In this work, we investigated the role of miR773 in modulating resistance to infection by fungal pathogens in Arabidopsis thaliana. Interference with miR773 activity by target mimics (in MIM773 plants) and concomitant upregulation of the miR773 target gene METHYLTRANSFERASE 2 (MET2) increased resistance to infection by necrotrophic (Plectosphaerrella cucumerina) and hemibiotrophic (Fusarium oxysporum, Colletototrichum higginianum) fungal pathogens. By contrast, both MIR773 overexpression and MET2 silencing enhanced susceptibility to pathogen infection. Upon pathogen challenge, MIM773 plants accumulated higher levels of callose and reactive oxygen species than wild-type plants. Stronger induction of defense-gene expression was also observed in MIM773 plants in response to fungal infection. Expression analysis revealed an important reduction in miR773 accumulation in rosette leaves of plants upon elicitor perception and pathogen infection. Taken together, our results show not only that miR773 mediates pathogen-associated molecular pattern-triggered immunity but also demonstrate that suppression of miR773 activity is an effective approach to improve disease resistance in Arabidopsis plants.

MiR858-Mediated Regulation of Flavonoid-Specific MYB Transcription Factor Genes Controls Resistance to Pathogen Infection in Arabidopsis.

MicroRNAs (miRNAs) are a class of short endogenous non-coding small RNAs that direct post-transcriptional gene silencing in eukaryotes. In plants, the expression of a large number of miRNAs has been shown to be regulated during pathogen infection. However, the functional role of the majority of these pathogen-regulated miRNAs has not been elucidated. In this work, we investigated the role of Arabidopsis miR858 in the defense response of Arabidopsis plants to infection by fungal pathogens with necrotrophic (Plectosphaerella cucumerina) or hemibiotrophic (Fusarium oxysporum and Colletotrichum higginsianum) lifestyles. Whereas overexpression of MIR858 enhances susceptibility to pathogen infection, interference with miR858 activity by target mimics (MIM858 plants) results in disease resistance. Upon pathogen challenge, stronger activation of the defense genes PDF1.2 and PR4 occurs in MIM858 plants than in wild-type plants, whereas pathogen infection induced weaker activation of these genes in MIR858 overexpressor plants. Reduced miR858 activity, and concomitant up-regulation of miR858 target genes, in MIM858 plants, also leads to accumulation of flavonoids in Arabidopsis leaves. The antifungal activity of phenylpropanoid compounds, including flavonoids, is presented. Furthermore, pathogen infection or treatment with fungal elicitors is accompanied by a gradual decrease in MIR858 expression in wild-type plants, suggesting that miR858 plays a role in PAMP (pathogen-associated molecular pattern)-triggered immunity. These data support that miR858 is a negative regulator of Arabidopsis immunity and provide new insights into the relevant role of miR858-mediated regulation of the phenylpropanoid biosynthetic pathway in controlling Arabidopsis immunity.

Two NRAMP6 Isoforms Function as Iron and Manganese Transporters and Contribute to Disease Resistance in Rice.

Metal ions are essential elements for all living organisms. However, metals can be toxic when present in excess. In plants, metal homeostasis is partly achieved through the function of metal transporters, including the diverse natural resistance-associated macrophage proteins (NRAMP). Among them, the OsNramp6 gene encodes a previously uncharacterized member of the rice NRAMP family that undergoes alternative splicing to produce different NRAMP6 proteins. In this work, we determined the metal transport activity and biological role of the full-length and the shortest NRAMP6 proteins (l-NRAMP6 and s-NRAMP6, respectively). Both l-NRAMP6 and s-NRAMP6 are plasma membrane-localized proteins that function as iron and manganese transporters. The expression of l-Nramp6 and s-Nramp6 is regulated during infection with the fungal pathogen Magnaporthe oryzae, albeit with different kinetics. Rice plants grown under high iron supply show stronger induction of rice defense genes and enhanced resistance to M. oryzae infection. Also, loss of function of OsNramp6 results in enhanced resistance to M. oryzae, supporting the idea that OsNramp6 negatively regulates rice immunity. Furthermore, nramp6 plants showed reduced biomass, pointing to a role of OsNramp6 in plant growth. A better understanding of OsNramp6-mediated mechanisms underlying disease resistance in rice will help in developing appropriate strategies for crop protection.

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.

Salt-responsive ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice.

Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified salt-responsive ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance-mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK kinase kinase6 (MAP3K6), MAPK5, dehydration-responsive element bindinG2A (DREB2A), and zinc finger protein179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species-activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance.

Overexpression of a Calcium-Dependent Protein Kinase Confers Salt and Drought Tolerance in Rice by Preventing Membrane Lipid Peroxidation.

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

MicroRNA-mediated regulation of gene expression in the response of rice plants to fungal elicitors.

MicroRNAs (miRNAs) are small non-coding RNAs that have important regulatory functions in plant growth, development, and response to abiotic stress. Increasing evidence also supports that plant miRNAs contribute to immune responses to pathogens. Here, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs in rice. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection.

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors.

BACKGROUND: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity. Most studies on pathogen-regulated sRNAs have been conducted in Arabidopsis plants infected with the bacterial pathogen Pseudomonas syringae, or treated with the flagelin-derived elicitor peptide flg22 from P. syringae. This work investigates sRNAs that are regulated by elicitors from the fungus Fusarium oxysporum in Arabidopsis. RESULTS: Microarray analysis revealed alterations on the accumulation of a set of sRNAs in response to elicitor treatment, including miRNAs and small RNA sequences derived from massively parallel signature sequencing. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. Promoter analysis in transgenic Arabidopsis plants revealed transcriptional activation of MIR168 by fungal elicitors. Furthermore, transgenic plants expressing a GFP-miR168 sensor gene confirmed that the elicitor-induced miR168 is active. MiR823, targeting Chromomethylase3 (CMT3) involved in RNA-directed DNA methylation (RdDM) was also found to be regulated by fungal elicitors. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA. Studies on Arabidopsis mutants impaired in small RNA biogenesis demonstrated that this sRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. Hc-siRNAs are known to be involved in RNA-directed DNA methylation (RdDM). SiRNA415 is detected in several plant species. CONCLUSION: Results here presented support a transcriptional regulatory mechanism underlying MIR168 expression. This finding highlights the importance of miRNA functioning in adaptive processes of Arabidopsis plants to fungal infection. The results of this study also lay a foundation for the involvement of RdDM processes through the activity of siRNA415 and miR823 in mediating regulation of immune responses in Arabidopsis plants.

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.

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue.

In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4-2.5 g of glucose; and 0.73-2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.

Identification of a novel microRNA (miRNA) from rice that targets an alternatively spliced transcript of the Nramp6 (Natural resistance-associated macrophage protein 6) gene involved in pathogen resistance.

Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

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.

Development and Genome-Wide Analysis of a Blast-Resistant japonica Rice Variety.

Rice is one of the most important crops in the world, and its production is severely affected by the rice blast disease caused by the fungus Magnaporthe oryzae. Several major blast resistance genes and QTLs associated with blast resistance have been described and mostly identified in indica rice varieties. In this work, we report the obtention of a blast-resistant rice breeding line derived from crosses between the resistant indica variety CT13432 and the japonica elite cultivar JSendra (highly susceptible to blast). The breeding line, named COPSEMAR9, was found to exhibit resistance to leaf blast and panicle blast, as demonstrated by disease assays under controlled and field conditions. Furthermore, a high-quality genome sequence of the blast-resistant breeding line was obtained using a strategy that combines short-read sequencing (Illumina sequencing) and long-read sequencing (Pacbio sequencing). The use of a whole-genome approach allowed the fine mapping of DNA regions of indica and japonica origin present in the COPSEMAR9 genome and the identification of parental gene regions potentially contributing to blast resistance in the breeding line. Rice blast resistance genes (including Pi33 derived from the resistant parent) and defense-related genes in the genome of COPSEMAR9 were identified. Whole-genome analyses also revealed the presence of microRNAs (miRNAs) with a known function in the rice response to M. oryzae infection in COPSEMAR9, which might also contribute to its phenotype of blast resistance. From this study, the genomic information and analysis methods provide valuable knowledge that will be useful in breeding programs for blast resistance in japonica rice cultivars.

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.

Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection.

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.