Volume 4, Issue 5, Pages 909-921 (September 2011) Myosin VIII Regulates Protonemal Patterning and Developmental Timing in the Moss Physcomitrella patens  Wu Shu-Zon , Ritchie Julie A. , Pan Ai-Hong , Quatrano Ralph S. , Bezanilla Magdalena   Molecular Plant  Volume 4, Issue 5, Pages 909-921 (September 2011) DOI: 10.1093/mp/ssr068 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 P. patens Has Five Highly Similar Myosin VIII Genes.Gene models and conserved protein domain structures of P. patens myosin VIII genes are shown. Insertional knockouts were generated by homologous recombination. The knockout constructs are shown below each gene model. Black boxes denote exons that encode for protein, while gray boxes denote untranslated regions. Horizontal arrows above each gene model indicate primers used in RT–PCR for validating altered transcript in Δmyo8ABCDE (see Figure 5). Black lines under each gene model indicate sequence within the myo8 RNAi construct (see Figure 3). Protein sequences are highly similar among the five myosin VIII genes. All five myosin VIIIs have an N-terminal extension on the motor domain (black) and four IQ motifs (red) downstream of the motor domain. Two regions of coiled-coil (blue) are downstream of the IQ motifs. The vertical arrows above the protein diagrams indicate where protein translation is interrupted in the knockouts. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 All Myosin VIII Genes Are Expressed in Protonemata and Gametophores. Relative expression levels of myosin VIII genes normalized to ubiquitin 10 as determined by qRT–PCR with RNA isolated from 7–9-day-old protonemal tissue (dark gray) or from gametophores isolated from 2-month-old plants. Error bars represent standard error of the mean for at least two biological replicates. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 RNAi of Myosin VIII Results in Smaller Plants. (A) Representative fluorescence images of 6-day-old plants transformed with control RNAi or myo8–RNAi plasmids. Plants were stained with calcofluor and visualized by epifluorescence stereomicroscopy. Number of plants analyzed are: control RNAi n = 188, myo8–RNAi n = 101.(B) The average area normalized to the control RNAi is shown in the bar chart. Error bars represent standard error of the mean. Scale bar = 100 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Myosin VIII Is Not Required for Plant Viability. Representative fluorescence images of 6-day-old wild-type and myosin VIII knockout plants regenerated from protoplasts. Plants were stained with calcofluor and visualized by epifluorescence stereomicroscopy. Number of plants analyzed are 100 for all lines except: wild-type (n = 199), Δmyo8D (n = 98), Δmyo8BD (n = 101), Δmyo8BCDE (n = 99), Δmyo8ABCDE (n = 297). The average area normalized to wild-type is shown in the bar chart for each line. Error bars indicate standard error of the mean. Scale bar = 100 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Myosin VIII Transcripts Are Altered in Δmyo8ABCDE.RT–PCR on RNA isolated from wild-type and Δmyo8ABCDE protonemal tissue with primers flanking the insertion regions of the resistant cassettes (shown in Figure 1). In Δmyo8ABCDE, no transcript for myo8A, myo8B, and myo8E was detected. Myo8C and myo8D transcripts are still present, but are 221 and 491 bp bigger, respectively, than the wild-type products. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Myosin VIII Is Involved in Branch Formation. Wild-type and Δmyo8ABCDE plants regenerated from protoplasts were grown on PpNH4 medium.(A) Brightfield images of protonemal filaments of 20-day-old plants. Arrows indicate branches forming on branches in Δmyo8ABCDE plants. Scale bar = 100 μm.(B) 12-day-old plants stained with calcofluor visualized by epifluorescence microscopy. Scale bar = 20 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Myosin VIII Controls the Rate of Development.(A) 20-day-old wild-type and Δmyo8ABCDE plants regenerated from single protoplasts were grown on PpNH4 medium. Δmyo8ABCDE plant contains fully developed gametophores while the same-aged wild-type plant has no visible gametophores. Scale bar = 1 mm.(B) 12-day-old wild-type and Δmyo8ABCDE plants grown from single protoplasts. Protoplasts are on PRM for 4 d, then grown on water containing agar for 4 d before transfer to water agar containing 10 μM cytokinin (6-BAP) for another 4 d. Scale bar = 100 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 Myosin VIII Is Required for Normal Growth in Limiting Nutrients. Wild-type and Δmyo8ABCDE plants regenerated from protoplasts plated in liquid media onto PRM for 4 d then transferred to water containing agar.(A) Top: 12-day-old plants. Scale bar = 1 mm. Bottom: The edge of 13-day-old plants. Scale bar = 1 mm.(B) Curvature index was quantified for protonemal filaments from 13-day-old plants. Curvature index for wild-type (n = 62) and Δmyo8ABCDE (n = 147) was plotted as a histogram. Inset shows the average curvature index. Error bar represents standard error of the mean.(C) Top: The edge of 13-day-old plants is shown. Filaments grew in an undulating pattern (arrows) in Δmyo8ABCDE. Bottom: The center of 13-day-old plants is shown. Branches were significantly shorter and unevenly distributed (arrow heads) along the main filaments in Δmyo8ABCDE. Scale bar = 100 μm.(D) 12-day-old plants stained with calcofluor visualized by epifluorescence stereomicroscopy. Scale bar = 20 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 Auxin Partially Rescues Morphological Defects in Δmyo8ABCDE. Wild-type and Δmyo8ABCDE plants were regenerated from protoplasts and plated in liquid media. Four days after protoplasting, plants were transferred to water containing agar. Four days later, they were transferred to water with or without 1 μM NAA. Scale bar = 100 μm. Molecular Plant 2011 4, 909-921DOI: (10.1093/mp/ssr068) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions