Reply: Future Challenge in Plant Multivesicular Body Studies

Vesicle trafficking is a large and complex research area, still in its infancy in plants. The Letter to the Editor in The Plant Cell by Robinson et al. (2011) is written by longtime experts in this area and makes a sincere and honest effort to clarify that “ARF1 localizes to the Golgi and the TGN. ” The letter is a comment on our recent article “The Multivesicular Body-Localized GTPase ARFA1b/1c Is Important for Callose Deposition and ROR2 SyntaxinDependent Preinvasive Basal Defense in Barley” published in The Plant Cell (Böhlenius et al., 2010), which questions the subcellular location of ARF1. Therefore, the letter is well justified. Robinson et al. suggest that our ARF GTPase localization to multivesicular bodies (MVBs) is erroneous, perhaps due to overexpression from a strong promoter, and that no ARF GTPases have been associated with MVBs, since no coatmediated vesicle budding occurs on this cellular compartment. This explanation is certainly possible. However, we remain open to the possibility that our result could be due to differences between dicots and monocots, or, perhaps more likely, that differences in both location and function may exist within the ARFA subfamily. Robinson et al. present an example that illustrates how colocalization can be misleading. However, the example involves two integral membrane proteins (syntaxin, SYP61, and vacuolar sorting receptor [VSR2]), both of which pass through the ER-Golgi pathway before reaching their final separate destinations. Therefore, it is not surprising that they colocalize for a period of time after transformation. This may not be a fully representative example to illustrate how an ARF (barley [Hordeum vulgare] ARFA1b/1c) and a Rab GTPase, both of which are expressed in the cytosol and subsequently bind to a particular membrane, might be mislocalized. Despite very few amino acid differences, barley ARFA1b/1c falls into a distinct group relative to the ARF1 group that includes barley ARFA1a and the Arabidopsis thaliana ARFA1 GTPases, as shown in Figure 1 of Böhlenius et al. (2010). Support for a functional difference between these two ARFA1 subgroups came initially from transient transformation of barley epidermal cells with constructs for At-ARFA1c-T31N and At-ARFA1c-Q71L. In these experiments, which we did not describe in Böhlenius et al. (2010), we observed no effect on penetration resistance to the barley powdery mildew fungus (108 and 101% penetration relative to control, respectively) unlike after the transformation with constructs for Hv-ARFA1b/1c-T31N and Hv-ARFA1b/1c-Q71L. This lack of effect of ARF1 subgroup proteins is supported by the Hv-ARFA1a RNA interference result in Figure 2A of Böhlenius et al. (2010). Robinson et al. suggest that the colocalization of ARFA1b/1c and ARA7, observed in Figure 7 of Böhlenius et al. (2010), may indicate that ARA7 has been shifted from MVBs to the trans-Golgi network as a result of the overexpression of ARFA1b/1c. This may, in fact, suggest a more plausible explanation for the function for ARFA1b/1c than the one we previously gave. Interference with a barley ARF GTPase associated with TGN to MVB trafficking, a suggested function for ARF1 in Arabidopsis (KleineVehn et al., 2008), could have a direct negative impact on MVBs and obstruct their delivery of ROR2 to the papillae, for instance, in accordance with our observations in Figure 4 of Böhlenius et al. (2010). The othologous syntaxin of ROR2 in Arabidopsis, PEN1, has previously been suggested to be transported via MVB intraluminal vesicles to exosomes in the papillae (Meyer et al., 2009). In conclusion, we believe that this discussion points to a need for more detailed examination of the role of ARF1 GTPases, and the associated ARF-GEF(s), in MVB formation and in localization of MVB markers, such as ARA6 and ARA7. Furthermore, it remains unclear whether the different members of the ARFA1 subfamily have separate functions despite their very close structural similarity. We believe that our data suggest separate functions, at least in monocots.