Marina Franceschetti

A Dual Upstream Open Reading Frame-based Autoregulatory Circuit Controlling Polyamine-responsive Translation

A novel form of translational regulation is described for the key polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC). Plant AdoMetDC mRNA 5′ leaders contain two highly conserved overlapping upstream open reading frames (uORFs): the 5′ tiny and 3′ small uORFs. We demonstrate that the small uORF-encoded peptide is responsible for constitutively repressing downstream translation of the AdoMetDC proenzyme ORF in the absence of increased polyamine levels. This first example of a sequence-dependent uORF to be described in plants is also functional in Saccharomyces cerevisiae. The tiny uORF is required for normal polyamine-responsive AdoMetDC mRNA translation, and we propose that this is achieved by control of ribosomal recognition of the occluded small uORF, either by ribosomal leaky scanning or by programmed -1 frameshifting. In vitro expression demonstrated that both the tiny and the small uORFs are translated. This tiny/small uORF configuration is highly conserved from moss to Arabidopsis thaliana, and a more diverged tiny/small uORF arrangement is found in the AdoMetDC mRNA 5′ leader of the single-celled green alga Chlamydomonas reinhardtii, indicating an ancient origin for the uORFs.

Polyamine biosynthetic diversity in plants and algae

Polyamine biosynthesis in plants differs from other eukaryotes because of the contribution of genes from the cyanobacterial ancestor of the chloroplast. Plants possess an additional biosynthetic route for putrescine formation from arginine, consisting of the enzymes arginine decarboxylase, agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase, derived from the cyanobacterial ancestor. They also synthesize an unusual tetraamine, thermospermine, that has important developmental roles and which is evolutionarily more ancient than spermine in plants and algae. Single-celled green algae have lost the arginine route and are dependent, like other eukaryotes, on putrescine biosynthesis from the ornithine. Some plants like Arabidopsis thaliana and the moss Physcomitrella patens have lost ornithine decarboxylase and are thus dependent on the arginine route. With its dependence on the arginine route, and the pivotal role of thermospermine in growth and development, Arabidopsis represents the most specifically plant mode of polyamine biosynthesis amongst eukaryotes. A number of plants and algae are also able to synthesize unusual polyamines such as norspermidine, norspermine and longer polyamines, and biosynthesis of these amines likely depends on novel aminopropyltransferases similar to thermospermine synthase, with relaxed substrate specificity. Plants have a rich repertoire of polyamine-based secondary metabolites, including alkaloids and hydroxycinnamic amides, and a number of polyamine-acylating enzymes have been recently characterised. With the genetic tools available for Arabidopsis and other model plants and algae, and the increasing capabilities of comparative genomics, the biological roles of polyamines can now be addressed across the plant evolutionary lineage.

Chitosan treatment induces changes of protein expression profile and stilbene distribution in Vitis vinifera cell suspensions

Polyphenols, including stilbenes and flavonoids, are an essential part of human diet and constitute one of the most abundant and ubiquitous groups of plant secondary metabolites, and their level is inducible by stress, fungal attack or biotic and abiotic elicitors. Proteomic analysis of Vitis vinifera (L.) cultivar (cv.) Barbera grape cell suspensions, showed that the amount of 73 proteins consistently changed in 50 μg/mL chitosan‐treated samples compared with controls, or between the two controls, of which 56 were identified by MS analyses. In particular, de‐novo synthesis and/or accumulation of stilbene synthase proteins were promoted by chitosan which also stimulated trans‐resveratrol endogenous accumulation and decreased its release into the culture medium. No influence was shown on cis‐resveratrol. There was no effect on the accumulation of total resveratrol mono‐glucosides (trans‐ and cis‐piceid and trans‐ and cis‐resveratroloside). Throughout the observation period the upregulation of phenylalanine ammonia lyase, chalcone synthase, chalcone‐flavanone isomerase (CHI) transcript expression levels well correlated with CHI protein amount and with the accumulation of anthocyanins. Chitosan treatment strongly increased the expression of eleven proteins of the pathogenesis related protein‐10 family, as well as their mRNA levels.

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001

Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) flowers and stalks collected from plants germinated and grown under increasing salt-stress conditions (0-75 mM NaCl). The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Spermidine synthase enzyme activity determined both in supernatant and pellet fractions, together with RT-PCR results, led us to hypothesize a different intracellular compartmentation of the isoforms of these enzymes. Free and conjugated polyamines (perchloric acid-soluble and -insoluble) were measured. Free spermidine was the most abundant polyamine and its levels, such as those of free spermine, increased with salt concentration, supporting the hypothesis for a specific role of those polyamines in the response and tolerance to salt stress of Arabidopsis thaliana flowers.

Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3′End Processing in Magnaporthe oryzae

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3′UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3′end processing mechanisms exclusive to the fungal kingdom.

Common Genetic Pathways Regulate Organ-Specific Infection-Related Development in the Rice Blast Fungus

This study describes fungal infection–related development of Magnaporthe oryzae induced on rice roots and on hydrophilic polystyrene. A fungal mutant with abnormal preinfection hyphae and lacking the ortholog of the karyopherin exportin-5 had defects in full disease symptom production on leaves and roots, showing that this fungal karyopherin plays an important role during plant colonisation. Magnaporthe oryzae is the most important fungal pathogen of rice (Oryza sativa). Under laboratory conditions, it is able to colonize both aerial and underground plant organs using different mechanisms. Here, we characterize an infection-related development in M. oryzae produced on hydrophilic polystyrene (PHIL-PS) and on roots. We show that fungal spores develop preinvasive hyphae (pre-IH) from hyphopodia (root penetration structures) or germ tubes and that pre-IH also enter root cells. Changes in fungal cell wall structure accompanying pre-IH are seen on both artificial and root surfaces. Using characterized mutants, we show that the PMK1 (for pathogenicity mitogen-activated protein kinase 1) pathway is required for pre-IH development. Twenty mutants with altered pre-IH differentiation on PHIL-PS identified from an insertional library of 2885 M. oryzae T-DNA transformants were found to be defective in pathogenicity. The phenotypic analysis of these mutants revealed that appressorium, hyphopodium, and pre-IH formation are genetically linked fungal developmental processes. We further characterized one of these mutants, M1373, which lacked the M. oryzae ortholog of exportin-5/Msn5p (EXP5). Mutants lacking EXP5 were much less virulent on roots, suggesting an important involvement of proteins and/or RNAs transported by EXP5 during M. oryzae root infection.

Single Amino Acid Mutations in the Potato Immune Receptor R3a Expand Response to Phytophthora Effectors

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR or NLR) proteins to respond to invading pathogens and activate immune responses. How plant NB-LRR proteins respond to pathogens is poorly understood. We undertook a gain-of-function random mutagenesis screen of the potato NB-LRR immune receptor R3a to study how this protein responds to the effector protein AVR3a from the oomycete pathogen Phytophthora infestans. R3a response can be extended to the stealthy AVR3aEM isoform of the effector while retaining recognition of AVR3aKI. Each one of eight single amino acid mutations is sufficient to expand the R3a response to AVR3aEM and other AVR3a variants. These mutations occur across the R3a protein, from the N terminus to different regions of the LRR domain. Further characterization of these R3a mutants revealed that at least one of them was sensitized, exhibiting a stronger response than the wild-type R3a protein to AVR3aKI. Remarkably, the N336Y mutation, near the R3a nucleotide-binding pocket, conferred response to the effector protein PcAVR3a4 from the vegetable pathogen P. capsici. This work contributes to understanding how NB-LRR receptor specificity can be modulated. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit.

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Translational regulation of the plant S-adenosylmethionine decarboxylase

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.