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

MicroRNAs in rice innate immunity

MicroRNAs (miRNAs) are short regulatory non-coding RNAs that guide gene silencing in most eukaryotes. They regulate gene expression by triggering sequence-specific cleavage or translational repression of target transcripts. Plant miRNAs are known to play important roles in a wide range of developmental processes. Increasing evidence also supports that the modulation of miRNA levels plays an important role in reprogramming plant responses to abiotic stress (drought, cold, salinity and nutrient deficiency) and biotic stress (antibacterial resistance). Most of these studies were carried out in the model plant Arabidopsis thaliana. During the last years, the adoption of high-throughput sequencing technologies has significantly contributed to uncover multiple miRNAs while allowing miRNA profiling in plants. However, although a plethora of rice miRNAs have been shown to be regulated by pathogen infection, the biological function remains largely unknown for most of them. In this review, we summarize our current understanding on the contribution of miRNAs to rice immunity and discuss their potential applications in rice biotechnology. A better understanding of the miRNA species controlling rice immunity may lead to practical biotechnological applications leading to the development of appropriate strategies for rice protection

A rice calcium-dependent protein kinase is expressed in cortical root cells during the presymbiotic phase of the arbuscular mycorrhizal symbiosis

<p>Abstract</p> <p>Background</p> <p>The arbuscular mycorrhizal (AM) symbiosis consists of a mutualistic relationship between soil fungi and roots of most plant species. This association provides the arbuscular mycorrhizal fungus with sugars while the fungus improves the uptake of water and mineral nutrients in the host plant. Then, the establishment of the arbuscular mycorrhizal (AM) symbiosis requires the fine tuning of host gene expression for recognition and accommodation of the fungal symbiont. In plants, calcium plays a key role as second messenger during developmental processes and responses to environmental stimuli. Even though calcium transients are known to occur in host cells during the AM symbiosis, the decoding of the calcium signal and the molecular events downstream are only poorly understood.</p> <p>Results</p> <p>The expression of seventeen Calcium-dependent Protein Kinase (CPK) genes representative of the four distinct phylogenetic groups of rice CPKs was monitored during the presymbiotic phase of the AM symbiosis. Among them, <it>OsCPK18 </it>and <it>OsCPK4</it>, were found to be transcriptionally activated in response to inoculation with the AM fungus <it>Glomus intraradices. </it><it>OsCPK18 </it>and <it>OsCPK4 </it>gene expression was also up-regulated by fungal-produced diffusible molecules. Laser microdissection revealed expression of <it>OsCPK18 </it>in cortical cells, and not in epidermal cells of <it>G. intraradices</it>-inoculated rice roots, suggesting a preferential role of this gene in the root cortex. Moreover, a plasma membrane localization of OsCPK18 was observed by transient expression assays of green fluorescent protein-tagged OsCPK18 in onion epidermal cells. We also show that the myristoylation site of the OsCPK18 N-terminus is required for plasma membrane targeting.</p> <p>Conclusion</p> <p>The rapid activation of <it>OsCPK18 </it>expression in response to AM inoculation, its expression being also induced by fungal-secreted signals, together with the observed plasma membrane localization of OsCPK18, points to a role for OsCPK18 in perception of the AM fungus. The <it>OsCPK18 </it>gene might be considered as a marker for the presymbiotic phase of the symbiotic process. These findings provide a better understanding of the signaling mechanisms operating during the AM symbiosis and will greatly facilitate their molecular dissection.</p

Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress

3 pages, 3 figures.-- PMID: 12432039 [PubMed].1-Aminocyclopropane-1-carboxylate (ACC) oxidase enzyme catalyses the final step in ethylene biosynthesis, converting 1-aminocyclopropane-1-carboxylic acid to ethylene. A cDNA clone encoding an ACC oxidase, ST-ACO3, was isolated from potato (Solanum tuberosum L.) by differential screening of a Fusarium eumartii infected-tuber cDNA library. The deduced amino acid sequence exhibited similarity to other ACC oxidase proteins from several plants species. Northern blot analysis revealed that the ST-ACO3 mRNA level increased in potato tubers upon inoculation with F. eumartii, as well as after treatment with salicylic acid and indole-3-acetic acid, suggesting a cross-talk between different signalling pathways involved in the defence response of potato tubers against F. eumartii attack.This work was partially supported by the IFS (Sweden), CONICET, UNMDP, ANPCyT (Grant No. 01-09768), and FundacioÂn Antorchas (Argentina). MEZ and CT were recipients of a fellowship from CONICET and UNMDP, respectively.Peer reviewe

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

The Polycistronic miR166k-166h positively regulates rice immunity via post-transcriptional control of EIN2

MicroRNAs (miRNAs) are small RNAs acting as regulators of gene expression at the post-transcriptional level. In plants, most miRNAs are generated from independent transcriptional units, and only a few polycistronic miRNAs have been described. miR166 is a conserved miRNA in plants targeting the HD-ZIP III transcription factor genes. Here, we show that a polycistronic miRNA comprising two miR166 family members, miR166k and miR166h, functions as a positive regulator of rice immunity. Rice plants with activated MIR166k-166h expression showed enhanced resistance to infection by the fungal pathogens Magnaporthe oryzae and Fusarium fujikuroi, the causal agents of the rice blast and bakanae disease, respectively. Disease resistance in rice plants with activated MIR166k-166h expression was associated with a stronger expression of defense responses during pathogen infection. Stronger induction of MIR166k-166h expression occurred in resistant but not susceptible rice cultivars. Notably, the ethylene-insensitive 2 (EIN2) gene was identified as a novel target gene for miR166k. The regulatory role of the miR166h-166k polycistron on the newly identified target gene results from the activity of the miR166k-5p specie generated from the miR166k-166h precursor. Collectively, our findings support a role for miR166k-5p in rice immunity by controlling EIN2 expression. Because rice blast is one of the most destructive diseases of cultivated rice worldwide, unraveling miR166k-166h-mediated mechanisms underlying blast resistance could ultimately help in designing appropriate strategies for rice protection

MiR858-mediated regulation of flavonoid-specific MYB transcription factor genes controls resistance to pathogen infection in Arabidopsis

Altres ajuts: CERCA Generalitat de CatalunyaMicroRNAs (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

OsDCL1a activation impairs phytoalexin biosynthesis and compromises disease resistance in rice

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. 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

Imaging the invasion of rice roots by the bakanae agent Fusarium fujikuroi using a GFP-tagged isolate

Altres ajuts: Generalitat de Catalunya/CERCA Programme, AGER Foundation (Grant 2010-2369)Fusarium fujikuroi (teleomorph Gibberella fujikuroi) is the main seed-borne pathogen of rice, the causal agent of bakanae, a disease that in the last years has become of increasing economical concern in many Italian rice growing areas. A virulent F. fujikuroi isolate was tagged with the green fluorescent protein (gfp) gene using Agrobacterium tumefaciens mediated transformation, and the virulence of the GFP isolate has been confirmed. Little is known about the early interaction of the pathogen with its host, in this work fungal development during the F. fujikuroi/root interaction was analysed by Laser Scanning Confocal Microscopy (LSCM), by using the GFP isolate obtained. The infection of rice roots was investigated from 48 h to 8 days post-inoculation both in resistant and susceptible cultivars. Roots of resistant genotype seem to trigger a hypersensitive response at the infection site and LSCM analysis of root sections allowed the visualization of fungal growth within host tissues. Fungal growth occurred both in the resistant and the susceptible cultivar, even if it was less abundant in the resistant one. Expression analysis of Chitinase1, a gene involved in fungal pathogenesis, was investigated by qPCR on the F. fujikuroi infected rice roots. Chitinase1 expression increased greatly upon infection in the resistant cultivar Selenio

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