Regulatory Patterns of a Large Family of Defensin-Like Genes Expressed in Nodules of <i>Medicago truncatula</i>

<div><p>Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume <i>Medicago truncatula</i> is the nodule cysteine-rich (<i>NCR</i>) group of defensin-like (<i>DEFL</i>) genes. We used a custom Affymetrix microarray to catalog the expression changes of 566 <i>NCR</i>s at different stages of nodule development. Additionally, bacterial mutants were used to understand the importance of the rhizobial partners in induction of <i>NCR</i>s. Expression of early <i>NCR</i>s was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late <i>NCR</i>s were induced concomitantly with bacteroid development in the nodules. The induction of early and late <i>NCR</i>s was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of <i>NCR</i>s were required for promoter activity. These <i>cis</i>-element motifs were found to be unique to the <i>NCR</i> family among all annotated genes in the <i>M. truncatula</i> genome, although they contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.</p> </div

Differentially expressed <i>NCR</i>s in nodules with different inoculations.

<p>^aTime point of nodule harvest.</p>b<p><i>S. meliloti</i> strain used for inoculation.</p><p>^cNumber of positively and negatively differentially expressed <i>NCR</i>s in comparison to mock-inoculated roots at 0 dpi. The <i>NCR</i>s with log₂ fold-change >1 and with false discovery rate adjusted P<0.05 are classified as differentially expressed.</p

The five conserved motifs found in the upstream regions of NCRs.

a<p>The motifs were identified using MEME, had the most significant E-values of all motifs identified and are represented in more than half of the input NCR sequences.</p>b<p>Number of promoters in which the motif was present in NCR genes.</p

Expression profiles of <i>NCR</i>s.

<p>Expression values are log₂-transformed intensity values. A, Hierarchical clustering (Euclidian average) of 571 NCR<i>s</i> and 14 treatments. Columns 1, 2, 3, 4, and 5 are data for mock-inoculated roots at 0, 4, 7, 14, and 40 dpi, respectively; columns 6, 7, 10, and 12 are roots inoculated with <i>S. meliloti</i> mutants <i>nodC</i>, <i>exoY</i>, <i>bacA</i> and <i>nifH</i> at 14 dpi, respectively; and columns 8, 9, 11, 13, and 14 are roots inoculated with Sm1021 at 3, 4, 7, 14, and 40 dpi, respectively. Color scales representing signal intensities are shown at the bottom. B, Expression profiles of 346 early <i>NCR</i>s. C, Expression profiles of 79 late <i>NCR</i>s. The box and whisker plots represent five different groups of intensity values, the minimum of which is the lowest whisker, the 25% quartile is represented by the bottom box, the 50% quartile is indicated by the median line, the 75% quartile is represented by the top box, the maximum value is the highest whisker, and the outliers are represented by x’s.</p

Localization of expression of an <i>NCR</i> in Medicago nodules.

<p>A, GUS staining of a nodule section with the MtTC100321_s_at promoter:GUS construct. B, Detection of the antisense probe corresponding to MtTC100321_s_at in a 10-µm thick nodule section. C, Magnification of boxed area in B. Bars in A and B are 200 µm. Bar in C is 100 µm. I, meristematic zone; II, infection zone; II-III, intermediate zone; III, nitrogen fixation zone of the nodule. All nodules were harvested at 14 dpi with Sm1021.</p

Correlation of <i>NCR</i> expression between nodules induced by Sm1021 and corresponding developmental time point mutants.

<p>All <i>NCR</i>s with “present” calls between the two treatments plotted were used to generate the scatter plots. Linear regression plots with a significant correlation (R²) are shown. A, Comparison between nodules induced by <i>exoY</i> at 14 dpi and Sm1021 at 3 dpi. B, Comparison between nodules induced by <i>bacA</i> at 14 dpi and Sm1021 at 7 dpi. C, Comparison between nodules induced by <i>nifH</i> at 14 dpi and Sm1021 at 14 dpi.</p

Quantification of bacteria in nodule extracts by flow cytometry.

<p>The density plots are based on fluorescence (GFP) detected against the rhizobial cell volume (Forward Scatter or FSC) in nodules formed 14 dpi with <i>nifH</i> or Sm1021. Twenty biological replicates (nodules from 20 different plants) were used for each treatment. The t-test p-value was >0.95.</p

Promoter deletion assays.

<p>A, segments used for promoter deletion assays of gene corresponding to MtTC103606_at. 1, 2, 3, 4, and 5 are the conserved nodule motifs. The motifs found on the antisense strand are denoted as −2 and −3. The letters a through i designate the positions of elements P$ID1_01, P$ARF_Q2, P$PBF_Q2, P$PBF_01, P$DOF2_01,$AGL1_01, ELEMENT1GMLBC3, TTGTCTCTT, and CTCTTT, respectively. B, C, and D are transgenic nodules with constructs for GUS expression containing 1,000 bp upstream from the translation start site (segment 2) of genes corresponding to MtTC103606_at, MtTC95126_at, and MtTC100321_s_at, respectively. Nodules were stained at 14 dpi for GUS activity. Bars are 200 µm.</p

Venn diagrams of expressed Arabidopsis <i>DEFL</i>s in different treatments.

<p>A, Number of expressed <i>DEFL</i>s and <i>MEG</i>s in various tissues. B, Comparison of different gene discovery approaches for the number of identified expressed <i>DEFL</i>s by each technique.</p

Transcriptional fusion analysis of Arabidopsis <i>DEFL</i> promoter constructs in T4 transgenic seedlings expressing pSCR:<i>LhG4</i>/<i>OP</i>:GUS.

<p>Histochemical staining for GUS activity in 1-week-old seedlings transformed with promoter constructs of Arabidopsis <i>DEFLs</i>. A, At1g60989. B, At2g26020. C, At5g60553. D, At3g63360. E, At4g30074. F, At5g05598. Scale bar 10 µm.</p