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. 2012 Aug;159(4):1671-85.
doi: 10.1104/pp.112.195990. Epub 2012 May 31.

Transcriptional Responses Toward Diffusible Signals From Symbiotic Microbes Reveal MtNFP- And MtDMI3-dependent Reprogramming of Host Gene Expression by Arbuscular Mycorrhizal Fungal Lipochitooligosaccharides

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Transcriptional Responses Toward Diffusible Signals From Symbiotic Microbes Reveal MtNFP- And MtDMI3-dependent Reprogramming of Host Gene Expression by Arbuscular Mycorrhizal Fungal Lipochitooligosaccharides

Lisa F Czaja et al. Plant Physiol. .
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Abstract

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn't Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca(2+) signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

Figures

Figure 1.
Figure 1.
Myc- and Nod-LCO-mediated activation of the MtENOD11 promoter in wild-type roots. M. truncatula plantlets expressing a pMtENOD11-GUS fusion (Journet et al., 2001) were grown on slant agar plates for 5 d prior to a 6-h (A) or 24-h (B) incubation in Myc control, 10−8 m nsMyc-LCO, 10−7 m nsMyc-LCO, 10−8 m sMyc-LCO, 10−8 m Nod-LCO, and Nod control solution (arranged in this order from top to bottom). Subsequently, roots were assayed for GUS activity for 24 h. Closeups show approximately 2-mm root segments treated with 10−7 m nsMyc-LCOs (left), 10−8 m sMyc-LCOs (middle), and 10−8 m Nod LCOs (right) after a 6-h (A) or 24-h (B) incubation. Bars = 2 mm.
Figure 2.
Figure 2.
MtENOD11 expression in wild-type roots challenged with Myc- and Nod-LCOs. Expression of MtENOD11 is shown in M. truncatula wild-type roots after 6 h (gray bars) and 24 h (black bars) of incubation with 10−8 m sMyc-LCOs (sMyc), 10−7 m nsMyc-LCOs (nsMyc), a mixture of both (s/nsMyc), Myc control solution (Myc ctrl.), 10−8 m Nod-LCOs (Nod), and Nod control solution (Nod ctrl.). A and B display results of Medicago GeneChip hybridizations (Supplemental Table S1), while C and D show the results of real-time RT-PCR measurements. Here, the mean expression values of three biological replicates per treatment are shown. Error bars represent se.
Figure 3.
Figure 3.
Differential gene expression in wild-type roots challenged with Myc- and Nod-LCOs. This diagram shows the number of genes significantly up-regulated (presented in black) and down-regulated (presented in gray) in M. truncatula wild-type roots after 6 h (left side) or 24 h (right side) of incubation with 10−8 m sMyc-LCOs (sMyc-LCO), 10−7 m nsMyc-LCOs (nsMyc-LCO), a mixture of both (s/nsMyc-LCO), and 10−8 m Nod-LCOs (Nod-LCO). In each case, expression ratios were calculated in comparison with control solutions. The number of genes significantly up- or down-regulated at least 1.5-fold (black bars) or 2-fold (striped bars) at P < 0.05 is plotted.
Figure 4.
Figure 4.
Identification of genes coactivated by Myc- and Nod-LCOs in wild-type roots. The Venn diagrams visualize the coactivation of gene expression in M. truncatula wild-type roots treated with 10−8 m sMyc-LCOs (sMyc), 10−7 m nsMyc-LCOs (nsMyc), a mixture of both Myc-LCOs (s/nsMyc), and 10−8 m Nod-LCOs (Nod) for 6 h (A) and 24 h (B). Numbers indicate the number of genes significantly up-regulated at least 1.5-fold at P < 0.05.
Figure 5.
Figure 5.
Signaling-related genes specifically activated by Myc-LCOs in wild-type roots. A summary is shown of the subset of genes encoding transcription factors or signal-related proteins being rapidly activated in M. truncatula wild-type roots after a 6-h treatment with 10−8 m sMyc-LCOs (sMyc), 10−7 m nsMyc-LCOs (nsMyc), or a mixture of both (s/nsMyc). For comparison, gene expression in response to a 6-h 10−8 m Nod-LCO (Nod) treatment is shown. Gene expression is represented as a heat map, with shades of red representing up-regulation and shades of green indicating down-regulation. An at least 1.5-fold (P < 0.05) up-regulation is indicated by white circles. Expression data were scaled to the maximum of a 2.4-fold regulation using Genesis software (Sturn et al., 2002).
Figure 6.
Figure 6.
Myc-LCO-mediated gene expression in the wild type and in symbiotic mutants. The diagrams show the number of genes (Supplemental Table S6) at least 1.5-fold (P < 0.05) induced in M. truncatula wild-type roots after 6 h of incubation with any Myc-LCO (WT column in A) or sMyc-LCOs (sMyc), nsMyc-LCOs (nsMyc), and a mixture of both Myc-LCOs (s/nsMyc; WT columns in B). Note that none of these genes is activated by Nod-LCOs at the cutoffs mentioned. The number of genes induced by Myc-LCOs in the wild type that are still activated in MtNFP and MtDMI3 mutants are shown on the right side of the wild-type columns. Values in B do not add up to the values in A due to overlapping activation of transcription by different Myc-LCOs.
Figure 7.
Figure 7.
Verification of Myc-LCO-mediated gene expression in the M. truncatula wild type and two different symbiotic mutants via real-time RT-PCR. A summary is shown of a subset of genes differentially activated in M. truncatula wild-type, nfp-1, and dmi3 roots after a 6-h treatment with 10−8 m sMyc-LCOs (sMyc), 10−7 m nsMyc-LCOs (nsMyc), or a mixture of both (s/nsMyc). Log2-transformed gene expression ratios are represented as heat maps, with shades of red representing up-regulation and shades of green indicating down-regulation. Expression patterns obtained via GeneChip hybridizations (left side) correlate with expression profiles recorded by real-time RT-PCR (right side). Expression data were scaled to the maximum of a 4-fold regulation using Genesis software (Sturn et al., 2002).
Figure 8.
Figure 8.
Model for LCO-mediated gene expression leading to nodulation, AM formation, and root-branching stimulation. This model integrates transcriptional responses toward Myc-LCOs in MtNFP and MtDMI3 mutants (Fig. 6) with current knowledge of LCO-mediated signal transduction. In precontact signaling, the perception of diffusible Nod- and Myc-LCOs requires a CSP consisting of the Lyr1-type LysM domain receptor kinase MtNFP, the Leu-rich repeat receptor kinase MtDMI2, the cation channel MtDMI1, the calcium/calmodulin-dependent protein kinase MtDMI3, and the GRAS transcription factor MtNSP2 (Gough and Cullimore, 2011). While the GRAS transcription factor MtNSP1 is specifically required for nodulation (Smit et al., 2005), a corresponding protein related to AM formation has not yet been reported (indicated by a question mark). Currently unknown Lyk-type LysM domain receptor kinases (designated LYK?; Gough and Cullimore, 2011) form heterodimers with MtNFP during initial Nod- and Myc-LCO signal perception. In addition to their requirement during the presymbiotic phase of nodulation and AM formation, Myc- and Nod-LCOs stimulate root branching via the CSP (Maillet et al., 2011). Downstream of the CSP, different components are required for root-branching stimulation (Maillet et al., 2011).

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