Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering

EXO70C2 is a key regulatory factor for optimal tip growth of pollen

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollenspecific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes

Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

EXO70A2 Is Critical for Exocyst Complex Function in Pollen Development

A pollen-specific component of the exocyst, a protein complex regulating cellular secretion, plays an important role in pollen development and function in Arabidopsis. Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction

Dissecting a Hidden Gene Duplication: The <i>Arabidopsis thaliana SEC10</i> Locus

<div><p>Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in <i>Arabidopsis thaliana</i> mutants in <i>SEC10</i>, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the <i>Arabidopsis thaliana SEC10</i> (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, <i>SEC10a</i> and <i>SEC10b</i>, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both <i>SEC10</i> genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of <i>A. thaliana</i>. Moreover, we show that the use of the existing <i>A. thaliana</i> reference genome sequence as a guide for sequence assembly of new <i>Arabidopsis</i> accessions or related species has at least in some cases led to error propagation.</p></div

The revisited structure of the <i>SEC10</i> locus in <i>Arabidopsis thaliana</i>.

<p>The revisited arrangement of the <i>SEC10</i> locus (At5g12370) depicts <i>SEC10a</i>, <i>SEC10b</i>, and parts of two neighboring genes (At5g12360, At5g12380). Coding exons are shown as black boxed, 5′UTR as gray boxes, and 3′ UTR as white boxes. Arrows indicate the position and orientation of primers used for cloning of the <i>SEC10</i> locus in four overlapping parts (a-I, a-II, b-I and b-II; lines at the bottom represent the ranges of the cloned PCR products). The orange strip marks the region omitted from the reference sequence of the <i>A. thaliana</i> genome.</p

Insertional mutations in the <i>A. thaliana SEC10</i> locus do not affect viability.

<p>Insertional mutations in the <i>A. thaliana SEC10</i> locus do not affect viability.</p

Analysis of T-DNA insertional mutants in <i>SEC10</i> genes of <i>A. thaliana.</i>

<p>(<b>A</b>) Positions of T-DNA insertions and primers used for genotyping (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094077#pone-0094077-t001" target="_blank">Table 1</a>) are indicated by triangles or arrows, respectively. Numbers below genes indicate the exact position of each insertion (in bp counted from the start codon) and long arrows show the gene orientation. (<b>B</b>) Expression levels of <i>SEC10a</i> and <i>SEC10b</i> in young seedlings of mutant lines as analyzed by semi-quantitative RT-PCR. The expression level of the ACT7 gene was used as a control.</p

Evidence for <i>SEC10</i> gene tandem duplication.

<p>(<b>A</b>) Expected outcomes of diagnostic PCR with outward-facing primers “A” and “B” (gray and black arrows), specific to each end of the <i>SEC10</i> gene, for potential structures of the <i>SEC10</i> locus (a single gene or three variants of tandem gene duplication). The table on the right shows the expected presence or absence (+ or −) of PCR products using different primer combinations. (<b>B</b>) Results of PCR reactions according to (<b>A</b>) using A. thaliana Col-0 genomic DNA as a template.</p

Expression levels of <i>SEC10a</i> and <i>SEC10b i</i>n various tissues of <i>A. thaliana</i>.

<p>(<b>A</b>) Specifity of the PCR primers demonstrated on paralog-specific cDNAs (AK222187 for <i>SEC10a</i> and AY096638 for <i>SEC10b</i>; indicated above the line), using primer sets specific for <i>SEC10a</i> or <i>SEC10b</i> (indicated below the line as <i>a</i> or <i>b</i>, respectively). (<b>B</b>) Expression levels of <i>SEC10a</i> and <i>SEC10b</i> in various tissues as analyzed by semi-quantitative RT-PCR. The expression level of the <i>ACT7</i> gene was used as a control.</p