Abstract
Arbuscular mycorrhiza (AM) is a symbiosis between plants and AM fungi that requires the intracellular accommodation of the fungal partner in the host. For reciprocal nutrient exchange, AM fungi form intracellular arbuscules that are surrounded by the peri-arbuscular membrane. This membrane, together with the fungal plasma membrane, and the space in between, constitute the symbiotic interface, over which nutrients are exchanged. Intracellular establishment of AM fungi requires the VAPYRIN protein which is induced in colonized cells, and which localizes to numerous small mobile structures of unknown identity (Vapyrin-bodies). In order to characterize the identity and function of the Vapyrin-bodies we pursued a dual strategy. First, we co-expressed fluorescently tagged VAPYRIN with a range of subcellular marker proteins, and secondly, we employed biochemical tools to identify interacting partner proteins of VAPYRIN. As an important tool for the quantitative analysis of confocal microscopic data sets from co-expression of fluorescent proteins, we developed a semi-automated image analysis pipeline that allows for precise spatio-temporal quantification of protein co-localization and of the dynamics of organelle association from movies. Taken together, these experiments revealed that Vapyrin-bodies have an endosomal identity with trans-Golgi features, and that VAPYRIN interacts with a symbiotic R-SNARE of the VAMP721 family, that localizes to the same compartment.
Highlights
Arbuscular mycorrhiza (AM) represents a wide-spread symbiotic association of plants with a monophyletic group of fungal endosymbionts (AM fungi) that are collectively known as the Glomeromycotina in the order Mucuromycota (Spatafora et al, 2016)
In order to systematically explore the identity of the VAPYRINbodies, we sought for an amenable expression system in which VAPYRIN localization could be studied in combination with a diverse panel of subcellular marker proteins
Student’s pairwise t-test revealed that the differences in the average distances of the three pairs (Figure 5d) were highly significant (p < 0.001). These results suggest that Vapyrin-bodies have an identity that involves features of recycling endosomes and trans-Golgi network (TGN), and that they are associated with the Golgi apparatus
Summary
Arbuscular mycorrhiza (AM) represents a wide-spread symbiotic association of plants with a monophyletic group of fungal endosymbionts (AM fungi) that are collectively known as the Glomeromycotina in the order Mucuromycota (Spatafora et al, 2016). An exceptional case among the essential genes in AM is the VAPYRIN gene It has been discovered independently in two host species, Medicago truncatula and Petunia hybrida, in three research groups involving both, forward and reverse genetic strategies (Feddermann et al, 2010; Pumplin et al, 2010; Murray et al, 2011). AM fungal infection involves the accumulation of multiple exocytic markers, including Golgi stacks and vesicles, at sites of hyphal progression, indicating that intracellular accommodation of AM fungi requires active membrane dynamics (Genre et al, 2012). The default target membrane for secretion is the PAM that surrounds the fungal arbuscule and controls nutrient fluxes between both partners (Pumplin et al, 2012) Apart from this information, the identity and function of the Vapyrinbodies has remained largely elusive. Our results indicate that Vapyrinbodies have a mixed identity with trans-Golgi/endosomal characteristics which are compatible with a role in transport and secretion
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