Venus-XLG2 localises to the cell periphery in unchallenged

XLG2 localization studies in Nicotiana benthamiana confirmed previous studies (Chakravorty et al., 2015; Maruta et al., 2015) which found XLG2 to be localized to the nucleus, cytoplasm and plasma membrane. In order to investigate the subcellular localization of XLG2 in Arabidopsis thaliana, pXLG2::Venus-XLG2 was transformed into Col-0, agb1-2, Col-3 gl1 and cerk1-4 plants. Confocal laser scanning microscopy revealed localization of XLG2 to the cell periphery in unchallenged Col-0 plants. These results confirm the localization studies conducted with C-terminal GFP fusions in this study and are in contrast to a recent study with stably transformed Arabidopsis plants overexpressing GFP-XLG2 from the 35S promoter (Maruta et al., 2015). To address the question if XLG2 localization might be stimulus dependent, leaves of Col-0 plants expressing Venus-XLG2 were infiltrated with H₂O, chitin or flg22. Analysis by confocal laser scanning microscopy revealed that 3 hours after each of these treatments, the Venus-XLG2 fluorescence signal increased overall and a distinct signal within nuclei appeared. After one day of incubation, the Venus-XLG2 signal intensity was increased further, with pronounced labelling of nuclei. These data suggest that in the wild type background, infiltration stress causes Venus-XLG2 abundance to increase and triggers its accumulation in nuclei. No clear difference in the subcellular behaviour of Venus-XLG2 could be seen between water and PAMP treatment. To investigate if the accumulation of Venus-XLG2 in nuclei is specifically caused by infiltration, leaves of Col-0 plants expressing Venus-XLG2 were analyzed after wounding. Leaf discs were cut out and analyzed either directly by confocal laser scanning microscopy or stored in water for 3 and 24 hours, respectively. Similar to infiltration of H₂O, chitin or flg22, a Venus-XLG2 signal in the nucleus appeared 3 hours after wounding and became more intense after 24 hours Thus it seems likely that different types of abiotic and biotic stress can trigger nuclear accumulation of Venus-XLG2 (Figure 34, Figure 35, Figure 36, Figure 37)

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Figure 34. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after H2O infiltration in Col-0 plants. Stably transformed Col-0 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with H2O using a syringe. Leaf discs were either used for microscopy directly after infiltration or were incubated in H2O for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

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Figure 35. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after chitin infiltration in Col-0 plants. Stably transformed Col-0 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with 100 mg ml^-1 chitin using a syringe. Leaf discs were either used for microscopy directly after infiltration or were incubated in 100 mg ml^-1 chitin solution for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

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Figure 36. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after flg22 infiltration in Col-0 plants. Stably transformed Col-0 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with 100nm flg22 using a syringe. Leaf discs were either used for microscopy directly after infiltration or were incubated in 100nm flg22 solution for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

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Figure 37. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after wounding in Col-0 plants. Stably transformed Col-0 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and directly used for microscopy of were left in water for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

Since XLG2 is required for the formation of the cerk1-4 phenotype, the localization of Venus-XLG2 was also analyzed in the cerk1-4 mutant and the corresponding wild type control, Col-3 gl1. The situation was the same as observed in Col-0. Venus-XLG2 localized to the cell periphery in unchallenged plants and showed an increase in overall signal intensity as well as accumulation in the nucleus upon water infiltration (Figure 38, Figure 39). Interestingly, when expressed in agb1-2 plants, Venus-XLG2 was localized to the cell periphery as well as the nucleus even in unchallenged plants. Upon infiltration of water, the signal at the cell periphery did not increase much, but the signal intensity in nuclei became very strong after 3 and 24 hours (Figure 40). The localization of Venus-XLG2 appeared to be shifted towards the nucleus in agb1-2 mutants.

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Figure 38. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after H2O infiltration in Col-3 gl1 plants. Stably transformed Col-3 gl1 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with H2O using a syringe. Leaf discs were either used for microscopy directly after infiltration or were incubated in H2O for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

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Figure 39. XLG2 is localized to the cell periphery in unchallenged plants and appears in nuclei after H2O infiltration in cerk1-4 plants. Stably transformed cerk1-4 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with H2O using a syringe. Leaf discs were either used for microscopy directly after infiltration or were incubated in H2O for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

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Figure 40. XLG2 is localized to the cell periphery and nucleus in unchallenged and challenged agb1-2 plants. Stably transformed agb1-2 plants expressing Venus-XLG2 from the XLG2 promoter were analyzed by Confocal laser scanning microscopy. Leaf discs were cut out and vacuum-infiltrated with H2O using a syringe.

Leaf discs were either used for microscopy directly after infiltration or were incubated in H2O for the indicated time points. Images represent maximum projections of 10 single focal plane images taken 1 µm apart. White arrows denote nuclei. Size bar indicates 10 µm.

To complement confocal laser scanning microscopy analyses, membrane association of Venus-XLG2 was investigated by immunoblotting. To do so, microsomal fractions were prepared from untreated transgenic plants expressing Venus-XLG2 in the 0, agb1-2, Col-3 gl1 or cerk1-4 backgrounds. For all genotypes, immunoblot analysis using a GFP antibody revealed the presence of Venus-XLG2 in total extracts and in soluble fractions, but not in microsomal fractions for all genotypes (Figure 41). Since microscopy indicated a plasma membrane localization of Venus-XLG2, the membrane association of Venus-XLG2 might be disrupted by the extraction process. A CERK1 immunoblot was performed using the same samples to validate the identity of the prepared fractions. Full length CERK1 (75 kDa) is

108 membrane bound and can only be found in total extracts and microsomal fractions, whereas the CERK1 ectodomain (33 kDa) can be found in total extacts and soluble fractions (Figure 41) (Petutschnig et al., 2014).

Figure 41. Venus-XLG2 can be found in soluble protein fractions, but not in microsomes. Microsomes were prepared from leaves of transgenic plants expressing Venus-XLG2. Samples were analyzed in immunoblot using a GFP antibody to detect Venus-XLG2 and with a specific CERK1 antibody, to validate microsomal and soluble fractions. Samples which have been used for GFP and CERK1 immunoblot are identical. CBB, Coommassie Brilliant Blue (loading control). Tot, total extracts; sol, soluble;

mic, microsomal fraction.

3.2.5.4 XLG2 is localized to the nucleus in Bgh attacked and surrounding