Figure 3.

Whi3 is required for polarized and clustered localization of BNI1 and SPA2 mRNAs. (A and H) mRNAs were immunoprecipitated (RIP) in a strain that expresses Whi3-TAP using α-TAP antibody (A) or in strains labeled using GFP-trap (ChromoTek; H), and identified after RT-PCR. Wild type (untagged Whi3) was used for RIP as a negative control. Total RNA was purified from the lysates (whole cell extract) of indicated strains and amplified after RT-PCR to confirm the presence of target mRNAs as inputs. (B, C, and I) Indicated mRNAs (yellow) are localized at hyphal tips (B and I) or nascent branches (C, arrow). Blue, DNA. Cells are outlined in gray. (D) The number of BNI1 or SPA2 mRNAs were counted at each 5 µm distance from the tip of hyphae and converted to a percentage. Error bars indicate SEM. n > 39. (E) Histograms (percentage of the total number of mRNAs) of NND in wild type, whi3Δ, or BNI1-3m for BNI1 or SPA2 (n > 1,224). NND in wild type are significantly different from the mutants. P < 0.01 by KS test. bni1-3m is expressed on a plasmid and compared with the strain episomally expressing BNI1-mCherry. (F) The degree of mRNA clustering (see Materials and methods) was calculated only with inter-region hyphal images (at least 25 µm away from the tip, n > 32). *, P < 0.01 compared with wild type using a KS test. (G) Whi3 (or ΔpolyQ, green) and Bni1 (or -3m, magenta) were localized in the same cell grown for 16 h (30°C; z-projected). The images shown are representatives (n > 15). (J) Cells were grown at 30°C for 20 h. Bars: (B, C, G, and I) 5 µm; (J) 200 µm.