Plants expressing murine pro-apoptotic protein Bid do not have enhanced PCD

The purpose of this study was to explore whether plant programmed cell death (PCD) cascade can sense the presence of the animal-only BH3 protein Bid, a BCL-2 family protein known to play a regulatory role in the signaling cascade of animal apoptosis

Selection of candidate genes controlling veraison time in grapevine through integration of meta-QTL and transcriptomic data

Background High temperature during grape berry ripening impairs the quality of fruits and wines. Veraison time, which marks ripening onset, is a key factor for determining climatic conditions during berry ripening. Understanding its genetic control is crucial to successfully breed varieties more adapted to a changing climate. Quantitative trait loci (QTL) studies attempting to elucidate the genetic determinism of developmental stages in grapevine have identified wide genomic regions. Broad scale transcriptomic studies, by identifying sets of genes modulated during berry development and ripening, also highlighted a huge number of putative candidates. Results With the final aim of providing an overview about available information on the genetic control of grapevine veraison time, and prioritizing candidates, we applied a meta-QTL analysis for grapevine phenology-related traits and checked for co-localization of transcriptomic candidates. A consensus genetic map including 3130 markers anchored to the grapevine genome assembly was compiled starting from 39 genetic maps. Two thousand ninety-three QTLs from 47 QTL studies were projected onto the consensus map, providing a comprehensive overview about distribution of available QTLs and revealing extensive co-localization especially across phenology related traits. From 141 phenology related QTLs we generated 4 veraison meta-QTLs located on linkage group (LG) 1 and 2, and 13 additional meta-QTLs connected to the veraison time genetic control, among which the most relevant were located on LG 14, 16 and 18. Functional candidates in these intervals were inspected. Lastly, taking advantage of available transcriptomic datasets, expression data along berry development were integrated, in order to pinpoint among positional candidates, those differentially expressed across the veraison transition. Conclusion Integration of meta-QTLs analysis on available phenology related QTLs and data from transcriptomic dataset allowed to strongly reduce the number of candidate genes for the genetic control of the veraison transition, prioritizing a list of 272 genes, among which 78 involved in regulation of gene expression, signal transduction or development

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed

IDENTIFICATION OF CANDIDATE GENES INVOLVED IN NITRIC OXIDE SIGNALLING DURING HYPERSENSITIVE-CELL DEATH

ABSTRACT L\u2019ossido nitrico (NO) \ue8 una molecola ampiamente diffusa che regola vari aspetti della crescita della pianta, del suo sviluppo e delle risposte di stress. Diversi studi hanno dimostrato che l\u2019accumulo di NO ed il suo signaling giocano un ruolo importante nella difesa delle piante contro i patogeni. In particolare l\u2019NO gioca un ruolo cruciale nel mediare la risposta di difesa ipersensibile. Questo meccanismo di resistenza implica l\u2019attivazione di una morte cellulare programmata al sito di infezione che ha come obiettivo quello di bloccare l\u2019infezione e la diffusione del patogeno (Jones and Dangl, 2006). In questo contesto si \ue8 dimostrato che l\u2019NO lavora in modo sinergico con il perossido di idrogeno per indurre la morte cellulare ipersensibile. Nonostante le molte ricerche su questa via di signaling, i meccanismi molecolari con cui agisce l\u2019NO non sono ancora chiari. Per acquisire nuove conoscenze specifiche nella via di signaling dell\u2019NO che regola l\u2019attivazione della morte cellulare ipersensibile abbiamo ottimizzato e testato un sistema di fumigazione con NO che consente di trattare le piante con quantit\ue0 controllate di questo gas. In questo lavoro abbiamo innanzitutto effettuato una ulteriore ottimizzazione di questo sistema per mettere a punto delle condizioni in grado di indurre la morte cellulare in modo uniforme e riproducibile in un numero maggiore di piante (320) di Arabidopsis thaliana di 4 settimane con lo scopo finale di incrementare il numero di mutanti testati. Quindi, utilizzando queste nuove condizioni abbiamo testato 39225 semi di Arabidopsis mutagenizzati con ESM (M2). Complessivamente l\u2019analisi di questi mutanti e di quelli precedentemente testati (17,107) hanno permesso di recuperare complessivamente 30 mutanti che presentano un fenotipo riproducibile di almeno parziale compromissione nella risposta all\u2019NO. Questi mutanti preselezionati sono quindi stati infettati anche col patogeno per confermare una possibile compromissione anche della morte cellulare ipersensibile indotta da patogeno. Di questi, 14 mutanti infatti hanno mostrato anche una compromissione di questa risposta, probabilmente quindi a causa di qualche alterazione nel signaling dell\u2019NO. Questi candidati sono stati sottoposti anche ad ulteriori analisi genetiche per testare l\u2019ereditariet\ue0 del fenotipo mutante e per confermare che, data la loro ereditariet\ue0, rappresentano materiale adatto per poter effettuare l\u2019identificazione della mutazione mediante un approccio di deep sequencing. Nello specifico l\u2019ereditariet\ue0 \ue8 stata determinata nelle popolazioni F1 ed F2 ottenute mediante backcross. L\u2019analisi fenotipica di queste popolazioni ha dimostrato che il fenotipo di uno dei candidati \ue8 dovuto ad una mutazione dominante e questo mutante \ue8 stato quindi escluso da ulteriori studi. Al momento le popolazioni BC1F2 e le analisi di segregazione in queste popolazioni sono state effettuate per tre dei 14 mutanti identificati. Inoltre test di allelismo tra sei dei mutanti selezionati hanno dimostrato che due mutanti erano allelici. Questi due mutanti condividono in realt\ue0 lo stesso evento di mutagenesi originale, dimostrando cos\uec l\u2019affidabilit\ue0 della procedura di screening. Con l\u2019obiettivo di proporre una strategia per l\u2019identificazione della mutazione causale mediante l\u2019approccio di mapping by sequencing, pools di ricombinanti che mostrano resistenza sono stati generati e sequenziati con un sequenziatore NGS Illumina. Tuttavia il nostro primo tentativo di identificare la mutazione causale basato su sequenziamento di pools ottenuti in BC1F2 non ha fornito uno sbilanciamento nello SNP-Index pari alle attese (1) al sito della mutazione causale a causa probabilmente di una contaminazione da falsi positivi nel pool di sequenziamento. Tuttavia i risultati di questa analisi suggeriscono di utilizzare per la costruzione di pools per il sequenziamento ricombinanti propagati il cui fenotipo sia stato attentamente verificato in F3 per poter effettuare con successo l\u2019identificazione della mutazione causale con questo approccio.Nitric oxide (NO) is a widespread signalling molecule that regulates various aspects of plant growth, development and stress responses. Numerous studies have demonstrated NO accumulation and downstream NO signalling plays an important role in plant defence against pathogens. In particular NO plays a crucial role in mediating the hypersensitive disease resistance response (HR). This resistance mechanism includes the activation of a programmed cell death at the attempted sites of infection, aiming to restrict pathogen infection and spread (Jones and Dangl, 2006). NO was shown to work synergistically with hydrogen peroxide to trigger the HR-cell death. Despite extensive investigation on this signalling pathway, the molecular mechanism through which NO acts is still unclear. To gain further insights specifically into NO signaling network underlying the activation of HR-cell death, a NO fumigation system, which allows treating plants with a precise amount of NO gas concentration in air has been established in our laboratory and previously tested. An optimization of such system was then successfully applied here to establish conditions of NO treatment that activate a uniform and reproducible cell death program in a larger number (320) of 4-week-old Arabidopsis thaliana plants, with the final aim of improve the screening performance. Then by using this facility and newly established NO fumigation conditions we screened further 39225 M2 EMS mutagenized Arabidopsis thaliana. These, together with previously fumigated mutants, complexively allowed to rescue 30 mutants presenting a consistent impaired NO response phenotype. These pre-selected mutants were then infected by pathogen to confirm a possible alteration also in HR-cell death. Among these, 14 mutants were found to be impaired HR-cell death likely because of alterations related to NO-signalling. The candidates were then subjected to additional genetic analysis to test inheritance of the mutant phenotype and to confirm they were suitable for to the identification of the causal mutation through deep sequencing based strategies. The genetic inheritance of the mutant phenotype was determined through analysis in the backcross F1 and F2 progeny. Phenotypic evaluation of the BC1F1 progeny demonstrated that phenotype of one of the candidates is caused by a dominant mutation. Therefore, this candidate was excluded from further studies. So far, BC1F2 populations and segregation analysis in the F2 progeny have been performed for three of the 14 candidate mutants. Furthermore allelism among six selected mutants was checked which revealed two allelic mutants, which were however sharing the same original mutational event, thus strengthening the reliability of our screening procedure. In order to set a strategy for the identification of the causal mutation by the \u201cmapping by sequencing\u201d approach, sequencing pools of resistant BC1F2 recombinants were generated and sequenced by Illumina NGS. However, our first attempt to identify causal mutation based on bulked BC1F2 didn\u2019t provide expected SNP index at putative causal mutation loci because of false positive contamination in the sequencing pool, suggesting that BC1F3 recombinants with a confirmed phenotype should be used, in our case, for the identification of the causal mutation through this approach

Measurement of Hypersensitive Cell Death Triggered by Avirulent Bacterial Pathogens in Arabidopsis

The hypersensitive response is one of the most powerful and complex defense reactions to survive to pathogen attacks during an incompatible plant-pathogen interaction. Local programmed cell death accompanies the hypersensitive response at the site of infection to prevent pathogen growth and spread. A precise quantitative assessment of this form of programmed cell death is essential to unravel the genetic and molecular mechanisms underlying the process. Here, we first describe the optimization of a Trypan Blue staining protocol for quantitatively measuring the HR-cell death in Arabidopsis. Furthermore, we provide an electrolyte leakage protocol based on pathogen vacuum infiltration, which allows its simultaneous application to a large number of plants as well as to Arabidopsis mutants affected by small size phenotype

Host-MediatedS-Nitrosylation Disarms the Bacterial Effector HopAI1 to Reestablish Immunity

Pathogens deliver effectors into plant cells to suppress immunity-related signaling. However, effector recognition by the host elicits a hypersensitive response (HR) that overcomes the inhibition of host signaling networks, restoring disease resistance. Signaling components are shared between the pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity, and it is unclear how plants inactivate these effectors to execute the HR. Here, we report that, inArabidopsis thaliana, during the onset of the HR, the bacterial effector HopAI1 isS-nitrosylated and that this modification inhibits its phosphothreonine lyase activity. HopAI1 targets and suppresses mitogen-activated protein kinases (MAPKs). TheS-nitrosylation of HopAI1 restores MAPK signaling and is required during the HR for activation of the associated cell death.S-nitrosylation is therefore revealed here as a nitric oxide-dependent host strategy involved in plant immunity that works by directly disarming effector proteins

Nitric Oxide Signaling during the Hypersensitive Disease Resistance Response.

Nitric oxide (NO) signaling is known to play a key role in triggering the hypersensitive response (HR) of plants to avirulent pathogens. In this chapter, we have summarized what is currently known about the role of NO in this important biological context. We have discussed NO production and turnover leading to the accumulation of this reactive compound when plants are challenged by pathogens. Unfortunately, enzymatic system for its production and the molecular basis for its accumulation are still largely unknown. Furthermore, we have reviewed the ways by which NO transduces its activity to establish hypersensitive disease resistance response by discussing novel emerging findings about its functions. By considering characterized NO targets, we have shown that NO signaling mainly relies on its reactivity with diverse protein targets. This finally orchestrates the crosstalk of NO with other signaling pathways and modulates defence gene expression, allowing the execution of HR cell death and the triggering of the defence respons

Functional conservation of the grapevine candidate gene INNER NO OUTER for ovule development and seed formation.

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed