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. 2009 Nov 27;107(1):478–483. doi: 10.1073/pnas.0910081107

Fig. 4.

Fig. 4.

Silencing FLOTs results in a decrease in nodule number, a decrease in infection events, and nodules that do form are nonfunctional. (A) Expression of FLOT2, FLOT3, and FLOT4 in individual hairy roots expressing the indicated RNAi or amiRNA construct was assessed by qRT-PCR, normalized to an internal actin control, and then to expression in plants transformed with the empty vector (average of at least 10 roots). Constructs are designated by their primary target gene(s) (*, >50% reduction in expression); numbers in parentheses show genes that have partial but significant (#, P < 0.05) reduction in expression due to cross silencing. (B) Nodulation phenotypes were assayed from a minimum of three biological replicates representing 50–100 plants per construct. (C) Acetylene reduction assays were performed at 21 dpi to determine the efficacy of nodules that form (14-20 plants were assayed per silenced line). (D) Hairy roots were stained for Rm1021 expressing pHemA:lacZ and stained for lacZ activity at 7 dpi to visualize infection events. Infection events and nodules were scored on ten transformed plants per amiRNA or RNAi and normalized to root weight (Fig. S6D). Between 104 (FLOT2,3,4 RNAi) and 371 (empty vector) total infection events were observed per line. (E) ITs on silenced roots (from Fig. 4C) were scored as aborting in the root hair, penetrating the hypodermal/cortical cell layers but not releasing bacteria into cortical cells, or releasing bacteria into cortical cells. Release was inferred by a blue haze in the nodule cortex. (A–E) Statistical significance was determined by pairwise two-tailed t tests comparing silenced lines to the empty vector control (*#, P < 0.05). (F–I) Abortive ITs in plants transformed with the FLOT4 amiRNA construct (F and G) compared to control ITs (H and I). (Scale bars: 15 μm.) (J) Linear regressions were conducted on plants described in A to determine whether a correlation exists between expression of FLOTs and nodule number. FLOT2 expression level has a strong linear relationship with nodule number (y = 5.1x + 2.7; P intercept = 0.005; P slope = 3 × 10−5); FLOT4 expression level has a weaker but statistically significant correlation (y = 2.0x + 5.8; P intercept = 3 × 10−8; P slope = 0.02). FLOT3 expression does correlates with nodule number (P slope = 0.6). FLOT2 and FLOT4 expression have additive effects (y = 7.1x + 0.9; P intercept = 0.003; P slope = 3 × 10−10).