Functional analysis of arbuscular mycorrhiza-related membrane transporter and defensin genes of Medicago truncatula

The arbuscular mycorrhiza (AM) symbiosis, an interaction of ~80% of terrestrial plants and Glomeromycota fungi, dating back ~450 million years and promoting the colonization of land by plants, improves the nutrient supply of the host. Vice versa the mutualistic interaction itself is influenced by nutrient availability, which alters the cost-benefit-ratio. Thus, the expression of AM-related membrane transporter and selected marker genes was studied in Medicago truncatula roots, mycorrhized with Rhizophagus irregularis, supplied with different phosphate (P) and nitrogen (N) amounts. Here, the root colonization, as well as the rate of arbuscule formation, were enhanced by P depletion, implying a high impact of nutrient allocation on the symbiosis. More than 100 membrane transporter genes were induced in the first two weeks of the AM-interaction and their accumulated induction increased further during the next four weeks. Five representative candidate genes from highly AM-induced gene families, encoding copper (MtCopMd1), oligopeptide (MtOliMd1), ABC (MtABCG3), and nitrogen (MtAMT2;4) membrane transporters as well as a defensin (MtDefMd1) were investigated to study processes at the plant-fungal interface. Arbuscule-harboring cells displayed different spacio-temporal levels of MtCopMd1-, MtOliMd1-, and MtABCG3 promoter activities, indicating a diverging regulation during AM. In this context, MtCopMd1 and MtAMT2;4 promoters were also activated by a strong nitrogen depletion, whereas MtOliMd1 and MtABCG3 were solely expressed in a P-dependent manner. Furthermore, the selected membrane transporter genes were differentially repressed in mutant or RNAi knockdown roots, lacking key regulators of AM symbioses. In Medicago truncatula roots expressing artificial micro-RNAs that target MtOliMd1 and MtABCG3, MtRam1, encoding a key transcription factor regulating arbuscular branching was significantly less expressed. Contrasting this, a strong knock-down of MtAMT2;4 expression by RNA-interference did not change the transcription of selected AM marker genes. Nevertheless, MtAMT2;4 localization correlated with arbuscule structures. The heterologous expression of MtAMT2;4 in frog oocytes indicated that ammonia, rather than ammonium, might be the transported substrate. Finally, the expression of MtDefMd genes, encoding defensins with specific structural properties, were studied. Here, a core set of five defensin genes was induced over the time of fungal colonization of Medicago truncatula roots. MtDefMd1 and MtDefMd2 activation was placed relative to arbuscule formation and degradation, using mutants in key AM-activated regulator genes. Since cells with fully developed arbuscules displayed different levels of MtDefMd1 and MtDefMd2 promoter activities, they indicated an up-regulation towards later stages of arbuscule formation. Co-localization of an MtDefMd1-mGFP6 fusion with subcellular fluorescence markers revealed that this defensin is associated with arbuscules about to collapse, and ultimately is located in vacuolar compartments. Consecutively, two EXO70 genes, associated with vesicle targeting, were found to be repressed in MtDefMd1-overexpressing roots. By monitoring the expression and localization of different AM-related membrane transporter genes and their encoded gene products, this thesis provides novel insights on processes that occur in either active or degrading arbuscules. These are starting points for further studies of the cellular targeting towards symbiotic membranes during the biogenesis and in particular during the degradation of arbuscule structures

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

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

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

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

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

Relative expression of <i>MtDefMd1-4</i> and selected AM marker genes in <i>M</i>. <i>truncatula</i> roots in a time course of mycorrhization.

<p>Expression of <i>MtDefMd1-4</i>, the fungal <i>α</i>-tubulin gene <i>GiTubα</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref036" target="_blank">36</a>] as well as the <i>M</i>. <i>truncatula</i> AM marker genes <i>MtPt4</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref066" target="_blank">66</a>] and <i>MtMyb1</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref017" target="_blank">17</a>] is given in relation to the expression of <i>MtTefα</i>. Plant roots were harvested weekly from 7 to 42 days post inoculation with <i>R</i>. <i>irregularis</i>. Biological replicates were three pools of four plant roots per treatment. The standard deviation of the three biological replicates is given.</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