Cytological characterization and allelism testing of anther developmental mutants identified in a screen of maize male sterile lines.

Proper regulation of anther differentiation is crucial for producing functional pollen, and defects in or absence of any anther cell type result in male sterility. To deepen understanding of processes required to establish premeiotic cell fate and differentiation of somatic support cell layers a cytological screen of maize male-sterile mutants has been conducted which yielded 42 new mutants including 22 mutants with premeiotic cytological defects (increasing this class fivefold), 7 mutants with postmeiotic defects, and 13 mutants with irregular meiosis. Allelism tests with known and new mutants confirmed new alleles of four premeiotic developmental mutants, including two novel alleles of msca1 and single new alleles of ms32, ms8, and ocl4, and two alleles of the postmeiotic ms45. An allelic pair of newly described mutants was found. Premeiotic mutants are now classified into four categories: anther identity defects, abnormal anther structure, locular wall defects and premature degradation of cell layers, and/or microsporocyte collapse. The range of mutant phenotypic classes is discussed in comparison with developmental genetic investigation of anther development in rice and Arabidopsis to highlight similarities and differences between grasses and eudicots and within the grasses

Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae.

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging

Perturbation of protein function.

<p>Yeast expressing fusions of each of the indicated proteins to the C-terminus of Cdc12 were imaged to assess whether they perturb its function. Perturbation of Cdc12 function manifests as misshapen yeast cells and/or mislocalized Cdc12. The green fluorescent proteins show minimal perturbation; mKate2 and mKO2 show major perturbation; mTagBFP2 is intermediate. Brightness has been normalized separately for each image so it is not comparable from image to image.</p

Recommended fluorescent protein combinations for yeast imaging.

<p>Recommended fluorescent protein combinations for imaging in yeast are broken down by filter set (horizontal axis) and experimental requirement (vertical axis). All proteins mentioned here are available in yeast tagging vectors either from this paper or from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-Sheff1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-Young1" target="_blank">[9]</a> and most are available from Addgene. Recommended proteins are listed first, with alternatives given in parentheses. It is likely that iFP1.4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-Shu1" target="_blank">[22]</a> or iRFP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-Filonov1" target="_blank">[23]</a> can be used to image in the far-red (Cy5) channel, but this has not been tested in yeast. mWasabi is dimmer that EGFP or GFPγ, but is not excited at 405 nm, allowing it to be multiplexed with T-Sapphire <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-ZapataHommer1" target="_blank">[14]</a>.</p

Fluorescent Proteins Tested.

<p>λex and λem are the peak excitation and emission wavelengths of the fluorescent protein, respectively. QY is the quantum yield and EC the extinction coefficient in M⁻¹ cm⁻¹. Brightness is the product of QY and EC, divided by 1000. Data was taken from the literature and is not available for GFPγ or MaxGFP.</p

Brightness of green and red fluorescent proteins.

<p>Yeast expressing fusions of each of the optimized fluorescent proteins to the TDH3 protein were imaged, and the mean fluorescence of each strain was calculated. Data from each day was normalized to EGFP (for green proteins) or mCherry (red proteins) to compensate for day-to-day fluctuations in lamp brightness and detection efficiency. The measurement was repeated on at least three days and the mean and standard error for each strain is plotted. * indicates a protein significantly brighter than EGFP or mCherry as determined by a one-sided t-test with 5% significance threshold. A. Green fluorescent proteins. B. Red fluorescent proteins imaged with an mCherry filter set. C. Red fluorescent proteins imaged with a Cy3 filter set.</p

Schematic design of tagging plasmids.

<p>The overall design of these plasmids is identical to the pFA6a-link tagging plasmids previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902-Sheff1" target="_blank">[6]</a>. yoFP is one of the 24 yeast optimized proteins cloned here and S.M. is the yeast selectable marker, either <i>SpHis5</i>, <i>CaUra3</i>, or KanR. These tagging sequences can be amplified with the forward primer (gene-specific sequence)-GGTGACGGTGCTGGTTTA and reverse primer (gene-specific sequence)-TCGATGAATTCGAGCTCG. A complete list of plasmids constructed in this study is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067902#pone.0067902.s002" target="_blank">Table S2</a>.</p