Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima.

BACKGROUND: Apple production in Sweden and elsewhere is being threatened by the fungus, Neonectria ditissima, which causes a disease known as European canker. The disease can cause extensive damage and the removal of diseased wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. RESULTS: The original protocol from 2003 was upgraded to use enzymes currently available in the market for making protoplasts. The protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing the green fluorescent protein (GFP). CONCLUSIONS: A new genetic transformation protocol has been established and the inclusion of hydroxyurea in the buffer during the protoplast-generation step greatly increased the creation of knockout mutants via homologous recombination. Pathogenicity assays using the GFP-mutants showed that the mutants were able to infect the host and cause disease.

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis.

Tudor staphylococcal nuclease (TSN; also known as Tudor-SN, p100, or SND1) is a multifunctional, evolutionarily conserved regulator of gene expression, exhibiting cytoprotective activity in animals and plants and oncogenic activity in mammals. During stress, TSN stably associates with stress granules (SGs), in a poorly understood process. Here, we show that in the model plant Arabidopsis thaliana, TSN is an intrinsically disordered protein (IDP) acting as a scaffold for a large pool of other IDPs, enriched for conserved stress granule components as well as novel or plant-specific SG-localized proteins. While approximately 30% of TSN interactors are recruited to stress granules de novo upon stress perception, 70% form a protein-protein interaction network present before the onset of stress. Finally, we demonstrate that TSN and stress granule formation promote heat-induced activation of the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1), the plant orthologue of mammalian AMP-activated protein kinase (AMPK). Our results establish TSN as a docking platform for stress granule proteins, with an important role in stress signalling.

Combining transcriptomics and genetic linkage based information to identify candidate genes associated with Heterobasidion-resistance in Norway spruce.

The Heterobasidion annosum s.l species complex comprises the most damaging forest pathogens to Norway spruce. We revisited previously identified Quantitative Trait Loci (QTLs) related to Heterobasidion-resistance in Norway spruce to identify candidate genes associated with these QTLs. We identified 329 candidate genes associated with the resistance QTLs using a gene-based composite map for Pinaceae. To evaluate the transcriptional responses of these candidate genes to H. parviporum, we inoculated Norway spruce plants and sequenced the transcriptome of the interaction at 3 and 7 days post inoculation. Out of 298 expressed candidate genes 124 were differentially expressed between inoculation and wounding control treatment. Interestingly, PaNAC04 and two of its paralogs in the subgroup III-3 of the NAC family transcription factors were found to be associated with one of the QTLs and was also highly induced in response to H. parviporum. These genes are possibly involved in the regulation of biosynthesis of flavonoid compounds. Furthermore, several of the differentially expressed candidate genes were associated with the phenylpropanoid pathway including a phenylalanine ammonia-lyase, a cinnamoyl-CoA reductase, a caffeoyl-CoA O-methyltransferase and a PgMYB11-like transcription factor gene. Combining transcriptome and genetic linkage analyses can help identifying candidate genes for functional studies and molecular breeding in non-model species.

Chemical Screening Pipeline for Identification of Specific Plant Autophagy Modulators.

Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.

Transcriptome analysis of embryonic domains in Norway spruce reveals potential regulators of suspensor cell death.

The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryo-suspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Transcript profiling of two potato cultivars during glycoalkaloid-inducing treatments shows differential expression of genes in sterol and glycoalkaloid metabolism.

Steroidal glycoalkaloids (SGA) are sterol-derived neurotoxic defence substances present in several members of the Solanaceae. In the potato (Solanum tuberosum), high SGA levels may render tubers harmful for consumption. Tuber SGA levels depend on genetic factors, and can increase as a response to certain stresses and environmental conditions. To identify genes underlying the cultivar variation in tuber SGA levels, we investigated two potato cultivars differing in their SGA accumulation during wounding or light exposure; two known SGA-inducing treatments. Using microarray analysis coupled to sterol and SGA quantifications, we identified a small number of differentially expressed genes that were associated with increased SGA levels. Two of these genes, encoding distinct types of sterol Δ24-reductases, were by sense/antisense expression in transgenic potato plants shown to have differing roles in sterol and SGA metabolism. The results show that an increased SGA level in potato tubers during both wounding and light exposure is mediated by coordinated expression of a set of key genes in isoprenoid and steroid metabolism, and suggest that differences in this expression underlie cultivar variations in SGA levels. These results may find use within potato breeding and quality assessment.

Identification of Norway Spruce MYB-bHLH-WDR Transcription Factor Complex Members Linked to Regulation of the Flavonoid Pathway.

Transcription factors (TFs) forming MYB-bHLH-WDR complexes are known to regulate the biosynthesis of specialized metabolites in angiosperms through an intricate network. These specialized metabolites participate in a wide range of biological processes including plant growth, development, reproduction as well as in plant immunity. Studying the regulation of their biosynthesis is thus essential. While MYB (TFs) have been previously shown to control specialized metabolism (SM) in gymnosperms, the identity of their partners, in particular bHLH or WDR members, has not yet been revealed. To gain knowledge about MYB-bHLH-WDR transcription factor complexes in gymnosperms and their regulation of SW, we identified two bHLH homologs of AtTT8, six homologs of the MYB transcription factor AtTT2 and one WDR ortholog of AtTTG1 in Norway spruce. We investigated the expression levels of these genes in diverse tissues and upon treatments with various stimuli including methyl-salicylate, methyl-jasmonate, wounding or fungal inoculation. In addition, we also identified protein-protein interactions among different homologs of MYB, bHLH and WDR. Finally, we generated transgenic spruce cell lines overexpressing four of the Norway spruce AtTT2 homologs and observed differential regulation of genes in the flavonoid pathway and flavonoid contents.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

A genome-wide association study identifies genomic regions for virulence in the non-model organism Heterobasidion annosum s.s.

The dense single nucleotide polymorphisms (SNP) panels needed for genome wide association (GWA) studies have hitherto been expensive to establish and use on non-model organisms. To overcome this, we used a next generation sequencing approach to both establish SNPs and to determine genotypes. We conducted a GWA study on a fungal species, analysing the virulence of Heterobasidion annosum s.s., a necrotrophic pathogen, on its hosts Picea abies and Pinus sylvestris. From a set of 33,018 single nucleotide polymorphisms (SNP) in 23 haploid isolates, twelve SNP markers distributed on seven contigs were associated with virulence (P<0.0001). Four of the contigs harbour known virulence genes from other fungal pathogens and the remaining three harbour novel candidate genes. Two contigs link closely to virulence regions recognized previously by QTL mapping in the congeneric hybrid H. irregulare × H. occidentale. Our study demonstrates the efficiency of GWA studies for dissecting important complex traits of small populations of non-model haploid organisms with small genomes.

Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen.

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism.

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and ß forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Identification of quantitative trait loci affecting virulence in the basidiomycete Heterobasidion annosum s.l.

Identification of virulence factors of phytopathogens is important for the fundamental understanding of infection and disease progress in plants and for the development of control strategies. We have identified quantitative trait loci (QTL) for virulence on 1-year-old Pinus sylvestris and 2-year-old Picea abies seedlings and positioned them on a genetic linkage map of the necrotrophic phytopathogen Heterobasidion annosum sensu lato (s.l.), a major root rot pathogen on conifers. The virulence of 102 progeny isolates was analysed using two measurements: lesion lengths and fungal growth in sapwood from a cambial infection site. We found negative virulence effects of hybridization although this was contradicted on a winter-hardened spruce. On P. abies, both measurements identified several partially overlapping QTLs on linkage group (LG) 15 of significant logarithm of odds (LOD) values ranging from 2.31 to 3.85. On P. sylvestris, the lesion length measurement also identified a QTL (LOD 3.09) on LG 15. Moreover, QTLs on two separate smaller LGs, with peak LOD values of 2.78 and 4.58 were identified for fungal sapwood growth and lesion lengths, respectively. The QTL probably represent loci important for specific as well as general aspects of virulence on P. sylvestris and P. abies.