Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots
ABSTRACT: The Poplar transcriptome was analyzed in mycorrhizal root tips in contact with Laccaria bicolor for 2 weeks. During mycorrhization the roots were treated with either 250M-BM-5m ACC, 10nM JA or 500M-BM-5M SA and compared to untreated mycorrhiza or control roots without contact to L. bicolor. In addition the poplar mutants 35S::PttACO1 and 35S::Atetr1 were used We performed 27 hybridizations (NimbleGen) with samples derived from Populus tremula x Populus alba clone 717-1B4 control roots, untreated mycorrhiza, SA-treated mycorrhiza, ACC-treated mycorrhiza and JA-treated mycorrhiza (3 biological replicates each) as well as Populus tremula x Populus tremuloides T89 control roots, mycorrhiza, 35S::PttACO1 mycorrhiza and 35S::Atetr1-1 mycorrhiza (3 biological replicates). All samples were labeled with Cy3.
Project description:The Poplar transcriptome was analyzed in mycorrhizal root tips in contact with Laccaria bicolor for 2 weeks. During mycorrhization the roots were treated with either 250µm ACC, 10nM JA or 500µM SA and compared to untreated mycorrhiza or control roots without contact to L. bicolor. In addition the poplar mutants 35S::PttACO1 and 35S::Atetr1 were used We performed 27 hybridizations (NimbleGen) with samples derived from Populus tremula x Populus alba clone 717-1B4 control roots, untreated mycorrhiza, SA-treated mycorrhiza, ACC-treated mycorrhiza and JA-treated mycorrhiza (3 biological replicates each) as well as Populus tremula x Populus tremuloides T89 control roots, mycorrhiza, 35S::PttACO1 mycorrhiza and 35S::Atetr1-1 mycorrhiza (3 biological replicates). All samples were labeled with Cy3.
Project description:Ectomycorrhizal fungi, such as Laccaria bicolor, support forest growth and sustainability by providing growth-limiting nutrients to their plant host through a mutualistic symbiotic relationship with host roots. We have previously shown that the effector protein MiSSP7 (Mycorrhiza-induced Small Secreted Protein 7) encoded by L. bicolor is necessary for the establishment of symbiosis with host trees, although the mechanistic reasoning behind this role was unknown. We demonstrate here that MiSSP7 interacts with the host protein PtJAZ6, a negative regulator of jasmonic acid (JA)-induced gene regulation in Populus. As with other characterized JASMONATE ZIM-DOMAIN (JAZ) proteins, PtJAZ6 interacts with PtCOI1 in the presence of the JA mimic coronatine, and PtJAZ6 is degraded in plant tissues after JA treatment. The association between MiSSP7 and PtJAZ6 is able to protect PtJAZ6 from this JA-induced degradation. Furthermore, MiSSP7 is able to block--or mitigate--the impact of JA on L. bicolor colonization of host roots. We show that the loss of MiSSP7 production by L. bicolor can be complemented by transgenically varying the transcription of PtJAZ6 or through inhibition of JA-induced gene regulation. We conclude that L. bicolor, in contrast to arbuscular mycorrhizal fungi and biotrophic pathogens, promotes mutualism by blocking JA action through the interaction of MiSSP7 with PtJAZ6.
Project description:Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein-protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein-protein interaction network, maintaining the repression of PtMYC2.1-regulated genes.
Project description:The early phase of the interaction between tree roots and ectomycorrhizal (ECM) fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We set out to identify gene networks that regulate LR development during the early signal exchanges between Populus tremula x Populus alba (hereafter called poplar) and the ECM fungus Laccaria bicolor. A sandwich culture system was developed in order to bring plant and fungus into an indirect contact, which permits signal molecule exchange and LR stimulation (emergence after 4-5 days of contact) but prohibits root colonization. A NimbleGen full genome poplar oligo-array was used to investigate transcript profiles at three days of indirect poplar/L. bicolor contact, referring to the time point of LR initiation in response to the fungus. Overall design: The Populus whole-genome expression array version 2.0 (S. DiFazio, A. Brunner, P. Dharmawardhana, and K. Munn, unpublished data) manufactured by NimbleGen Systems Limited (Madison, WI) contains in duplicates three independent, non-identical, 60-mer probes per whole gene model plus control probes and labeling controls. Included in the microarray are 65,965 probe sets corresponding to 55,970 gene models predicted on the P. trichocarpa genome sequence version 1.0 and 9,995 aspen cDNA sequences (Populus tremula, Populus tremuloides, and P. tremula x P. tremuloides). NimbleGen whole genome microarray analyses were performed in triplicate as per manufacturers instructions. We carried out six hybridizations (NimbleGen) with samples derived from each six pooled, entire in vitro grown root systems on MES-buffered medium. Three samples (biological replicates) originated from control roots in the absence of L. bicolor (GSM415421, GSM417830, GSM417831) and another serie of three biological replicates consisted of roots from a three days indirect contact with L. bicolor (GSM417832, GSM417833, GSM417856). cDNA was synthesized using CLONTECH Smart cDNA Synthesis kit containing an amplification step on the cDNA level. All samples were labeled with Cy3.
Project description:Illumina GAIIx technology was used to generate mRNA profiles from the ectomycorrhizal fungi Laccaria bicolor colonizing roots of Populus trichocarpa. Samples were taken after two, four and 12 weeks of contact in order to identify mycorrhiza-regulated transcripts. 37bp reads were generated and aligned to the Populus trichocarpa (http://www.phytozome.net/poplar.php) and the Laccaria bicolor (http://genome.jgi-psf.org/Lacbi2/Lacbi2.home.html) reference genomes using CLC Genomics Workbench 6. mRNA profiles from Populus trichocarpa roots colonized by Laccaria bicolor for two, four and 12 weeks as well as from control roots and free-living mycelium were generated by using one lane of 37 bp Illumina GAIIx sequencing per sample.
Project description:This study characterizes the transcriptomic alterations of P. tremula x P. alba at three weeks after inoculation with the ectomycorrhizal fungus Laccaria bicolor. We performed 6 hybridizations (NimbleGen) with samples derived from Populus tremula x P. alba control roots and mycorrhizal root tips. Samples were taken after 3 weeks of interaction (three biological replicates). All samples were labeled with Cy3.
Project description:The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F(1) individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, ?-glucuronidase, cellobiohydrolase, ?-glucosidase, ?-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.
Project description:We used whole-genome microarrays to identify differentially expressed genes in leaves of GA-deficient (35S::PcGA2ox) and/or GA-insensitive (35S::rgl1) transgenics as compared to WT poplar (717-1B4 genotype). Our work suggests that the molecular machinery that reduces gibberellins (GAs) concentration and signaling is a major route for restraining growth under both immediate and imminent adverse conditions. We show that inhibition of growth as a result of water deprivation and short days (SDs) coincides with up-regulation of several DELLA and GA2ox encoding genes in poplar. Likewise, GA-deficient and GA-insensitive transgenics, with up-regulated GA2ox and DELLA domain proteins, elicited a hypersensitive growth inhibition in response to both drought and SDs. Because the GA-modified transgenic showed accelerated response to drought and SD, we hypothesized that the mechanisms associated with these responses are constitutively elevated even under control conditions (well-watered, long day photoperiod). Therefore, we used whole-genome poplar microarray to study transcriptome level changes in the leaves of transgenic compared to WT plants grown under control environment. Genetic background for all plants was INRA 717-1B clone (Populus tremula x Populus alba). Expression analysis was preformed on three individual genotypes; wild-type (WT, untransformed control), 35S::PcGA2ox and 35S::rgl1. Leaves from two independent biological replicates per genotype were used, each pooled from 20 clonally propagated plants.
Project description:Mycorrhiza helper bacteria (MHB) are known to increase host root colonization by mycorrhizal fungi but the molecular mechanisms and potential tripartite interactions are poorly understood. Through an effort to study Populus microbiome, we isolated 21 Pseudomonas strains from native Populus deltoides roots. These bacterial isolates were characterized and screened for MHB effectiveness on the Populus-Laccaria system. Two additional Pseudomonas strains (i.e., Pf-5 and BBc6R8) from existing collections were included for comparative purposes. We analyzed the effect of co-cultivation of these 23 individual Pseudomonas strains on Laccaria bicolor "S238N" growth rate, mycelial architecture and transcriptional changes. Nineteen of the 23 Pseudomonas strains tested had positive effects on L. bicolor S238N growth, as well as on mycelial architecture, with strains GM41 and GM18 having the most significant effect. Four of seven L. bicolor reporter genes, Tra1, Tectonin2, Gcn5, and Cipc1, thought to be regulated during the interaction with MHB strain BBc6R8, were induced or repressed, while interacting with Pseudomonas strains GM17, GM33, GM41, GM48, Pf-5, and BBc6R8. Strain GM41 promoted the highest roots colonization across three Populus species but most notably in P. deltoides, which is otherwise poorly colonized by L. bicolor. Here we report novel MHB strains isolated from native Populus that improve L. bicolor root colonization on Populus. This tripartite relationship could be exploited for Populus species/genotypes nursery production as a means of improving establishment and survival in marginal lands.
Project description:Illumina HiSeq2000 technology was used to generate mRNA profiles from the ectomycorrhizal fungi Laccaria bicolor colonizing roots of Populus trichocarpa. Samples were taken after 3 months of contact in order to identify mycorrhiza-regulated transcripts. 100bp reads were generated and aligned to the Populus trichocarpa (http://www.phytozome.net/poplar.php) reference genome. Overall design: mRNA profiles from Populus trichocarpa roots colonized by Laccaria bicolor for three months as well as from control roots were generated by using one lane of 1X100bp Illumina HiSeq2000 sequencing per sample.