A Golden Gate Modular Cloning Toolbox for Plants

Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant <i>Nicotiana benthamiana</i> is discussed

Taxol partially rescues MTs from XopL_Xe.

Confocal microscopy of lower epidermal cells of GFP-TUA6 (labels MTs) stable transgenic N. benthamiana leaves. Leaves were agroinfected (OD600 of 0.4) to express (A-C) XopLXe-mCherry, (D-F) XopLm-mCherry and (K-M) mCherry and treated with the MT stabilizing chemical taxol (solved in DMSO) at 4 hpi. Samples were harvested for microscopy 2 dpi. The GFP channel is visible in white (labeled MTs) and the mCherry channel in magenta. Plastids are in cyan; ‘n’ labels nuclei. Scale bars are 20 μm. (G), (H), (I), (J) Are magnified images from (A), (B), (D) and (E), respectively (area magnified is outline with a white box). Scale bars in (G-J) are 5 μm. Examples of MTs are labeled with white arrows. (TIFF)</p

MT association is correlated with MTs disassembly.

Linear regression comparing MT binding ability of XopLm derivatives with MT number remaining after expression of XopLXe derivatives. Each data point represents the MT association of a given E3 ligase mutant variant (graphed in Fig 5C) plotted against the MT number remaining after expression of the corresponding E3 ligase-active version (graphed in Fig 5D). Each data point is labeled with the derivative name. The line of best fit is blue, and the equation of the line is displayed in the upper right (Linear Regression, R2 = 0.762, F [1, 7] = 22.35, P = 0.002). (TIFF)</p

DMSO control infiltrations into XopL_Xe-expressing tissue did not rescue MTs.

Confocal microscopy of lower epidermal cells of GFP-TUA6 (labels MTs) stable transgenic N. benthamiana leaves. Leaves were agroinfected (OD600 of 0.4) to express (A-C) XopLXe-mCherry, (D-F) XopLm-mCherry and (K-M) mCherry which was then treated with DMSO at 4 hpi. Samples were harvested for microscopy 2 dpi. The GFP channel is visible in white (labeled MTs), the mCherry channel in magenta. Plastids are visible in cyan. Scale bars are 20 μm. (TIFF)</p

Clustal Omega multiple sequence alignment of select XopL homologs from the <i>Xanthomonas</i> genus.

XopL protein sequences from 24 strains were aligned. The strain of origin is listed on the left-hand side with the NCBI accession number of the XopL protein sequence. Amino acids are colored based on polarity (Geneious Prime). Acidic amino acids in red, basic in blue, and fuchsia highlights prolines. The XopLXe proline-rich region (PRR) is in gray, the alpha α region (α-helices 1, 2 and 3) in yellow, and the beginning of the LRR domain (visible as ‘LRR1’) in light pink. The sequence logo above the alignment shows sequence conservation at specific positions. (TIFF)</p

KTN1 and PHS1 destroy MTs.

Confocal microscopy of lower epidermal cells of GFP-TUA6 stable transgenic N. benthamiana leaves. Leaves were agroinfected (OD600 of 0.8) to express (A) mCherry, (B) KTN1, (C) PHS1 185-700aa (PHS185-700) or (D) PHS1 1-700aa (PHS1-700) tagged with GFP. Samples were harvested for microscopy at 2 dpi. Images show the GFP channel, where MTs (GFP-TUA6 labeled) are typically visible (i.e., panel A). Scale bars are 20 μm. (E-H) are zoomed in versions of (A-D) respectively. (I) Cell death quantification via red fluorescence scanning of agroinfected N. benthamiana leaves. Tissue co-expressing XopLXe (purple) or non-MT-binding derivatives (ex2127/130; blue and αLRR_XL; orange) together with GFP or MT-disrupting proteins KTN1 and PHS1185-700 was monitored for cell death 5 dpi. Boxes represent first to third quartiles, the median is marked by a horizontal line and whiskers show the distribution of remaining data points. Treatments that were significantly different than XopLXe+ GFP co-inoculations are marked with asterisks (* = p0.001; One Way Analysis of Variance on Ranks, Bonferroni post-hoc test). (TIFF)</p

XopL_Xoo and XopL_Xac localize to MTs.

Confocal microscopy of lower epidermal cells of GFP-TUA6 stable transgenic N. benthamiana leaves. Leaves were agroinfected (OD600 of 0.4) to express synthesized (codon-optimized) (A) XopLXac and (B) XopLXoo translationally fused to a C-terminal mCherry. (A) and (B) are zoomed-out versions of cells depicted in Fig 4G and Fig 4J, respectively. mCherry-tagged XopLs are visible in magenta and the GFP channel is not shown here, ‘n’ marks the nucleus, white arrows show example MTs. Scale bars are 20 μm. Insets are magnifications of the nuclei (scale bar is 10 μm). (C) Plant reactions to codon-optimized XopLs were monitored at 6 dpi. (TIFF)</p