Annotation of protein-coding genes is very important in bioinformatics and biology and has a decisive influence on many downstream analyses. Homology-based gene prediction programs allow for transferring knowledge about protein-coding genes from an annotated organism to an organism of interest.Here, we present a homology-based gene prediction program called GeMoMa. GeMoMa utilizes the conservation of intron positions within genes to predict related genes in other organisms. We assess the performance of GeMoMa and compare it with state-of-the-art competitors on plant and animal genomes using an extended best reciprocal hit approach. We find that GeMoMa often makes more precise predictions than its competitors yielding a substantially increased number of correct transcripts. Subsequently, we exemplarily validate GeMoMa predictions using Sanger sequencing. Finally, we use RNA-seq data to compare the predictions of homology-based gene prediction programs, and find again that GeMoMa performs well.Hence, we conclude that exploiting intron position conservation improves homology-based gene prediction, and we make GeMoMa freely available as command-line tool and Galaxy integration.

Xanthomonas campestris pv. vesicatoria type III-secreted effectors were screened for candidates influencing plant cell processes relevant to the formation and maintenance of stromules in Nicotiana benthamiana lower leaf epidermis. Transient expression of XopL, a unique type of E3 ubiquitin ligase, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus. Further characterization of XopL revealed that the E3 ligase activity is essential for the two plastid phenotypes. In contrast to the XopL wild type, a mutant XopL lacking E3 ligase activity specifically localized to microtubules. Interestingly, mutant XopL-labeled filaments frequently aligned with stromules, suggesting an important, yet unexplored, microtubule-stromule relationship. High time-resolution movies confirmed that microtubules provide a scaffold for stromule movement and contribute to stromule shape. Taken together, this study has defined two populations of stromules: microtubule-dependent stromules, which were found to move slower and persist longer, and microtubule-independent stromules, which move faster and are transient. Our results provide the basis for a new model of stromule dynamics including interactions with both actin and microtubules.

SUMMARY Genome editing by RNA‐guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron‐optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene‐free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter‐selection markers for N. benthamiana, most importantly Sl‐FAST2, comparable to the well‐established Arabidopsis seed fluorescence marker, and FCY‐UPP, based on the production of toxic 5‐fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY‐UPP for selection of non‐transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher‐order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.

BackgroundAgrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes.ResultsUsing a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation.ConclusionAlthough we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as ‘normal’ as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy.

SUMMARYThe formation of Arabidopsis leaf veins is believed to require canalization of auxin into discrete and continuous cell files to generate a highly reproducible branched and reticulate pattern. During canalization, incipient veins become preferred routes for auxin transport through expression and asymmetric localization of the PINFORMED1 (PIN1) auxin efflux protein: PIN1 expression narrows from a group of cells to a single cell file, and localization of PIN1 protein becomes polarized to the cell membrane facing a previously formed vein. The shift in PIN1 localization is believed to require active vesicle cycling and be auxin-dependent, generating an autoregulatory loop. Previously, we have shown that fkd1 mutant leaves have an open vein pattern that lacks distal vein meeting. Here, we identify FKD1 as encoding a pleckstrin homology domain-and DUF828-containing protein. A fusion of the FKD1 promoter and the GUS reporter gene was expressed in vascular tissue throughout the plant, and its expression in incipient veins in leaves narrows in a manner similar to that of PIN1. FKD1 expression in roots and leaves can be altered by changes to auxin response and auxin transport. In the absence of FKD1, PIN1::GFP narrowing to incipient veins is delayed, and localization to the apical cell face is infrequent. The lack of apical PIN1 localization correlates with the failure of newly forming veins to connect distally with previously formed veins. Our data suggest that FKD1 influences PIN1 localization in an auxindependent manner, and we propose that it represents a key component of the auxin canalization pathway.