Sequential reprogramming of Medicago truncatula root cells during the accomodation of symbiotic arbuscular mycorrhizal fungi

[no abstract

A roadmap of cell-type specific gene expression during sequential stages of the arbuscular mycorrhiza symbiosis

BACKGROUND: About 80% of today’s land plants are able to establish an arbuscular mycorrhizal (AM) symbiosis with Glomeromycota fungi to improve their access to nutrients and water in the soil. On the molecular level, the development of AM symbioses is only partly understood, due to the asynchronous development of the microsymbionts in the host roots. Although many genes specifically activated during fungal colonization have been identified, genome-wide information on the exact place and time point of their activation remains limited. RESULTS: In this study, we relied on a combination of laser-microdissection and the use of Medicago GeneChips to perform a genome-wide analysis of transcription patterns in defined cell-types of Medicago truncatula roots mycorrhized with Glomus intraradices. To cover major stages of AM development, we harvested cells at 5-6 and at 21 days post inoculation (dpi). Early developmental stages of the AM symbiosis were analysed by monitoring gene expression in appressorial and non-appressorial areas from roots harbouring infection units at 5-6 dpi. Here, the use of laser-microdissection for the first time enabled the targeted harvest of those sites, where fungal hyphae first penetrate the root. Circumventing contamination with developing arbuscules, we were able to specifically detect gene expression related to early infection events. To cover the late stages of AM formation, we studied arbusculated cells, cortical cells colonized by intraradical hyphae, and epidermal cells from mature mycorrhizal roots at 21 dpi. Taken together, the cell-specific expression patterns of 18014 genes were revealed, including 1392 genes whose transcription was influenced by mycorrhizal colonization at different stages, namely the pre-contact phase, the infection of roots via fungal appressoria, the subsequent colonization of the cortex by fungal hyphae, and finally the formation of arbuscules. Our cellular expression patterns identified distinct groups of AM-activated genes governing the sequential reprogramming of host roots towards an accommodation of microsymbionts, including 42 AM-activated transcription factor genes. CONCLUSIONS: Our genome-wide analysis provides novel information on the cell-specific activity of AM-activated genes during both early and late stages of AM development, together revealing the road map of fine-tuned adjustments of transcript accumulation within root tissues during AM fungal colonization

Pre-announcement of symbiotic guests: transcriptional reprogramming by mycorrhizal lipochitooligosaccharides shows a strict co-dependency on the GRAS transcription factors NSP1 and RAM1

BACKGROUND: More than 80 % of all terrestrial plant species establish an arbuscular mycorrhiza (AM) symbiosis with Glomeromycota fungi. This plant-microbe interaction primarily improves phosphate uptake, but also supports nitrogen, mineral, and water aquisition. During the pre-contact stage, the AM symbiosis is controled by an exchange of diffusible factors from either partner. Amongst others, fungal signals were identified as a mix of sulfated and non-sulfated lipochitooligosaccharides (LCOs), being structurally related to rhizobial nodulation (Nod)-factor LCOs that in legumes induce the formation of nitrogen-fixing root nodules. LCO signals are transduced via a common symbiotic signaling pathway (CSSP) that activates a group of GRAS transcription factors (TFs). Using complex gene expression fingerprints as molecular phenotypes, this study primarily intended to shed light on the importance of the GRAS TFs NSP1 and RAM1 for LCO-activated gene expression during pre-symbiotic signaling. RESULTS: We investigated the genome-wide transcriptional responses in 5 days old primary roots of the Medicago truncatula wild type and four symbiotic mutants to a 6 h challenge with LCO signals supplied at 10(-7/-8) M. We were able to show that during the pre-symbiotic stage, sulfated Myc-, non-sulfated Myc-, and Nod-LCO-activated gene expression almost exclusively depends on the LysM receptor kinase NFP and is largely controled by the CSSP, although responses independent of this pathway exist. Our results show that downstream of the CSSP, gene expression activation by Myc-LCOs supplied at 10(-7/-8) M strictly required both the GRAS transcription factors RAM1 and NSP1, whereas those genes either co- or specifically activated by Nod-LCOs displayed a preferential NSP1-dependency. RAM1, a central regulator of root colonization by AM fungi, controled genes activated by non-sulfated Myc-LCOs during the pre-symbiotic stage that are also up-regulated in areas with early physical contact, e.g. hyphopodia and infecting hyphae; linking responses to externally applied LCOs with early root colonization. CONCLUSIONS: Since both RAM1 and NSP1 were essential for the pre-symbiotic transcriptional reprogramming by Myc-LCOs, we propose that downstream of the CSSP, these GRAS transcription factors act synergistically in the transduction of those diffusible signals that pre-announce the presence of symbiotic fungi.DFG/SPP1212/KU-1478/4-1DFG/SPP1212/KU-1478/4-4DFG/SPP1212/KU-1478/4-2DFG/SPP1212/KU-1478/4-

The mycorrhiza-dependent defensin MtDefMd1 of Medicago truncatula acts during the late restructuring stages of arbuscule-containing cells.

Different symbiotic and pathogenic plant-microbe interactions involve the production of cysteine-rich antimicrobial defensins. In Medicago truncatula, the expression of four MtDefMd genes, encoding arbuscular mycorrhiza-dependent defensins containing an N-terminal signal peptide and exhibiting some differences to non-symbiotic defensins, raised over the time of fungal colonization. Whereas the MtDefMd1 and MtDefMd2 promoters were inactive in cells containing young arbuscules, cells with fully developed arbuscules displayed different levels of promoter activities, indicating an up-regulation towards later stages of arbuscule formation. MtDefMd1 and MtDefMd2 expression was absent or strongly down-regulated in mycorrhized ram1-1 and pt4-2 mutants, known for defects in arbuscule branching or premature arbuscule degeneration, respectively. A ~97% knock-down of MtDefMd1/MtDefMd2 expression did not significantly affect arbuscule size. Although overexpression of MtDefMd1 in arbuscule-containing cells led to an up-regulation of MtRam1, encoding a key transcriptional regulator of arbuscule formation, no morphological changes were evident. Co-localization of an MtDefMd1-mGFP6 fusion with additional, subcellular markers revealed that this defensin is associated with arbuscules in later stages of their life-cycle. MtDefMd1-mGFP6 was detected in cells with older arbuscules about to collapse, and ultimately in vacuolar compartments. Comparisons with mycorrhized roots expressing a tonoplast marker indicated that MtDefMd1 acts during late restructuring processes of arbuscule-containing cells, upon their transition into a post-symbiotic state

Sequence analyses of of AM-dependent defensins MtDefMd1-4 and AM-unrelated defensins.

<p>Secondary structures of MtDefMd1-4 and AM-unrelated defensin-like proteins of <i>M</i>. <i>truncatula</i> (A), a representation of their three-dimensional structures (B and C), as well as surface electrostatics (D and E) are shown. Predicted signal peptides were removed from the mature amino acid sequences. Consecutively, the defensins were aligned based on their secondary structures. Background colorisation of the amino acids (in A) indicate hydrophobicity in a scale from red to blue (red: high hydrophobicity). A conserved aspartic acid in the C-terminal region of MtDefMds is marked with a grey triangel. For the bi-domain defensin MtDef5, the domains MtDef5A (including a 7 amino acid linker towards the MtDef5B domain) and MtDef5B are shown. After modelling the three-dimensional structures of the MtDefMd1-4, MtDef4 and MtDef5A/B defensins, they were visualized (B and C) and their surface electrostatics were calculated (D and E). The region congruent to the γ-core motif is indicated with arrows. The following proteins were used for comparisons in addition to MtDefMd1-4: MtDef5 A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref065" target="_blank">65</a>], MtDef5 B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref065" target="_blank">65</a>]and MtDef4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref030" target="_blank">30</a>].</p

Size distribution of arbuscules in mycorrhized <i>M</i>. <i>truncatula MtDefMd1</i>-overexpression, <i>MtDefMd1/2</i>-knock-down, and pPT4:<i>gusA</i>int controls roots.

<p>Arbuscules were sorted into one of eleven size categories. In total, the size distribution of arbuscules in pUbi:MtDefMd1-overexpression (647 arbuscules), pPt4:MtDefMd1-overexpression (509 arbuscules), RNAi:MtDefMd1/2-knock-down (625 arbuscules), and pPt4:<i>gusA</i>int control roots (529 arbuscules) is depicted in orange, blue, green, and grey, respectively. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. For each construct, three pools of root fragments, each pool being derived from four plants, were analysed. Depicted is the standard error of the mean.</p

Relative expression of MtDefMd1 and selected AM marker genes in mycorrhized MtDefMd1-overexpression and pPT4:<i>gusA</i>int-expressing transgenic control roots of <i>M</i>. <i>truncatula</i>.

<p>Transcript amounts are shown relative to <i>MtTefα</i> (A) and were additionally normalized by building a ratio to <i>MtPt4</i>-expression (B). Measurements of pPT4:MtDefMd1 overexpression roots are coloured in light grey, measurements of pUbi:MtDefMd1 overexpression roots in medium grey, and measurements of pPT4:<i>gusA</i>int control roots in dark grey. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. n = 12 biological replicates, depicted is the standard error of the mean. Statistical significance: * p≤0.05.</p

Relative expression of <i>MtDefMd</i> and selected AM marker genes in mycorrhized RNAi:MtDefMd1/2 and RNAi:<i>gusA</i>int transgenic control roots of <i>M</i>. <i>truncatula</i>.

<p>Transcript amounts are shown relative to <i>MtTefα</i> (A) and were additionally normalized by building a ratio to <i>MtPt4</i>-expression (B). Measurements from RNAi:MtDefMd1/2 roots are colored in light grey, corresponding RNAi:<i>gusA</i>int control measurements in dark grey. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. n = 12 biological replicates, depicted is the standard error of the mean. Statistical significances: _* p≤0.05, _** p≤0.005.</p

Histochemical localization of <i>MtDefMd1</i> and <i>MtDefMd2</i> promoter activities.

<p>Activities of <i>MtDefMd1</i> (A-D) and <i>MtDefMd2</i> (E-H) promoters were studied in transgenic, mycorrhized roots of <i>M</i>. <i>truncatula</i> A17 wild type (A, B, E, and F) and <i>pt4-2</i> roots (C, D, G, and H). Representative images of roots after 18 (A, B, E, and F) or 56 (C, D, G, and H) days post inoculation with <i>R</i>. <i>irregularis</i>. The GUS-stainings (A, C, E, and G) as well as the Alexa-WGA Fluor 488 stainings (B, D, F, and H) were performed over night. Septa are denoted by arrows. Abbreviations: w, cells with weak promoter activity; s, cells with strong promoter activity.</p