(A) GO-term enrichment analysis of Arabidopsis thaliana genes induced after 24 days growth in low Pi conditions. Based on RNA-Seq analysis of A. thaliana genes that were differentially expressed in Pi-deficient versus Pi-sufficient conditions (log2FC > 1, fdr < 0.05), Gene Ontology (GO) analysis showed that among the top 100 A. thaliana genes that were upregulated at 24 dpi under Pi-starvation conditions, nine were related to ‘cellular response to phosphate starvation’ (GO: 0016036). The color coding visually represents p value in each Go term box (Red box shows lower p value than yellow.).

(B) Analysis of regulatory mutants of the A. thaliana phosphate starvation response (PSR). Ct-mediated and phosphate starvation-dependent activation of both Pht1;2 and Pht1;3 was abrogated in phr1 phl1 plants. q-RT-PCR analysis revealed the reduction of Pht1;2 expression in phr1 phl1 mutant plants relative to Col-0 wild-type plants grown in low Pi media at 24 dpi. Expression levels are shown relative to the mean expression of plant actin. (Col-0 with Ct versus phr1 phl1 with Ct, two-tailed t test, p < 0.01).

(C) q-RT-PCR analysis revealed the reduction of Pht1;3 expression in phr1 phl1 mutant plants grown in low Pi media at 24 dpi. Expression levels are shown relative to the mean expression of plant actin. (Col-0 with Ct versus phr1 phl1 with Ct, two-tailed t test, p < 0.01).

(D) Translocation of 33P-labeled orthophosphate from Ct to A. thaliana shoots. Col-0 plants were grown on low Pi medium without (mock, n = 39) or with Ct (n = 40), phf1 mutants with Ct (n = 40), and phr1 phl1 double mutants with Ct (n = 40) in a two-compartment system. 33P was added to the hyphal compartment and after 17 days 33P -incorporation into shoots was measured by scintillation counting. Columns represent counting results in kBq 33P /g dry weight (DW) of individual plants. The dotted line shows the median level of 33P background counts from mock inoculations.

(E) At 4 days after roots were inoculated with Ct (4 dpi), Ct biomass in phr1 phl1 mutant plants was significantly higher than in Col-0 wild-type plants grown under low Pi conditions (Col-0 versus phr1 phl1, two-tailed t test, p < 0.05). To measure fungal biomass by qPCR, 500 ng RNA from infected plants was used to amplify the Ct actin fragment (CtACT) and RNA amounts were normalized to plant actin (AT3G18780).

(F) At 4 dpi, Ct biomass in mutant phf1 plants was significantly higher than in Col-0 plants grown under low Pi conditions (Col-0 versus phf1, two-tailed t test, p < 0.05).

(G) Histogram showing the diameter of Ct and Ci colonies at 2 and 6 days after mycelial plugs of these fungi were transferred to MS medium containing 50 μM (low) or 625 μM (high) KH 2 PO 4 . Colony sizes of Ct or Ci were not significantly different between high (+) and low Pi (-) conditions. Bars = SE.

(H) Schematic representation of the targeted replacement of secreted acid phosphatase gene CT04_08450 by homologous recombination. Flanking sequences upstream and downstream of the acid phosphatase gene were cloned into the T-DNA of binary vector pBIG4MRHrev adjoining the hygromycin (HYG) resistance cassette. The upstream and downstream flanking sequences of the incoming T-DNA undergo double cross-over homologous recombination with the target sequences, resulting in hygromycin-resistant mutants lacking the target gene.

(I) Confirmation of the gene replacement event in fungal transformants Ct_KO1 and Ct_KO2 by PCR analysis using primer pairs 1 and 2.

(J) Fungal growth in agar medium in which hydroxyapatite was the only P source. Both WT and the knockout strain Ct_KO1 were incubated for 3 days. Growth of the mutant was not significantly different to that of wild-type C. tofieldiae.