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

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

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

peer-reviewedThe 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

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

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.The Swedish Research Council Formas grant No. 2012–1276 and the Swedish Foundation for Strategic Research (SSF) grant No. R8b08-0011. This work was also supported by the National Science Foundation (IOS-1557796) to SM.http://www.frontiersin.orgam2017Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

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

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91191/1/j.1469-8137.2012.04128.x.pd

Heterobasidion root rot

Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.) is a necrotrophic pathogen causing damage to conifers in the Northern Hemisphere. H. annosum s.l. consists of five species: three European [H. annosum sensu stricto (s.s.), H. parviporum and H. abietinum] and two North American (H. irregulare and H. occidentale); all with different but partially overlapping host preferences. A multilocus phylogenetic tree was built and the divergence times were estimated. Plate tectonics is likely to have been the main factor influencing Heterobasidion speciation and biogeography. Along with the geographical separation, the Heterobasidion species have specialized on different host genera. The H. annosum species complex originated in Laurasia and the H. annosum s.s./H. irregulare and H. parviporum/H. abietinum/H. occidentale ancestral species emerged between 45 million–60 million years ago in the Palaearctic. The data imply that H. irregulare and H. occidentale colonized North America via different routes: H. irregulare colonizing from the east via Trans Atlantic land bridges and H. occidentale colonizing from the west via the Bering Land Bridge. Alternatively H. occidentale originated from North America. Identification of virulence factors is important for understanding the Heterobasidion–conifer pathosystem. Two studies of genetic mapping of virulence were performed. Virulence traits were measured as lesion length in the phloem and fungal growth in the sapwood of pine and spruce. Quantitative trait loci (QTL) were identified and positioned on a genetic linkage map for virulence of 102 progeny isolates from a cross between H. irregulare and H. occidentale. Both virulence traits in Picea abies identified significant QTLs on linkage group (LG) 15. Another QTL was positioned on LG 15 for the lesion length measurement in Pinus sylvestris. Moreover, QTLs on two separate smaller LGs were identified for fungal growth in sapwood and lesion length, respectively. The QTLs probably represent loci important for specific as well as general aspects of virulence on P. sylvestris and P. abies. A genome-wide association study was performed for virulence on 23 H. annosum s.s. isolates. Twelve SNP markers distributed on seven contigs were significantly associated with virulence. From these, three regions were characterized, two with one marker each with the lowest p-values and one region containing six markers. The linkage disequilibrium blocks in these regions ranged between 1.2 and 31.2 kb. Seven genes were identified as candidate virulence determinants encoding calcineurin, acetylglutamate kinase/synthase, cytochrome P450 monooxygenase, serine carboxypeptidase, quinone oxidoreductase (ToxD) and two flavin-containing monooxygenases

A Genome-Wide Association Study Identifies Genomic Regions for Virulence in the Non-Model Organism Heterobasidion annosum ss

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. irregulare6H. 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

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

Abstract 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\u2013196, 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\u2019H 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 Pa NAC03 and PaLAR3 in Nicotiana benthamiana showed that Pa NAC03 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