Cell detoxification of secondary metabolites by P4-ATPase-mediated vesicle transport

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1. 

   Version published to 10.7554/elife.79179 on eLife

   Jul 4, 2023
2. 

   eLife

   May 15, 2023

   Author Response

   Reviewer #1 (Public Review):

   This manuscript describes experiments that lead to a potentially impactful result and most of the data seem very nice. The authors conducted a mutant screen to find the gene BbCrpa from a fungus resistant to cyclosporine A (CsA). Microscopy indicates that the mode of action is likely sequestration of the toxin in vacuoles, mediated through the P4-ATPase pathway. They also show that expression of BbCrpa in Verticillium renders that fungus resistant to CsA. The paper then takes a very large jump across kingdoms and toxins and asks if BbCrpa, expressed in plants, will confer resistance to a different toxin (cinnamon acetate) that is produced by Verticillium. They conduct disease assays on Arabidopsis and cotton and show promising results, but these assays are less thoroughly completed. They …

   Author Response

   Reviewer #1 (Public Review):

   This manuscript describes experiments that lead to a potentially impactful result and most of the data seem very nice. The authors conducted a mutant screen to find the gene BbCrpa from a fungus resistant to cyclosporine A (CsA). Microscopy indicates that the mode of action is likely sequestration of the toxin in vacuoles, mediated through the P4-ATPase pathway. They also show that expression of BbCrpa in Verticillium renders that fungus resistant to CsA. The paper then takes a very large jump across kingdoms and toxins and asks if BbCrpa, expressed in plants, will confer resistance to a different toxin (cinnamon acetate) that is produced by Verticillium. They conduct disease assays on Arabidopsis and cotton and show promising results, but these assays are less thoroughly completed. They provide microscopic evidence that the transgenics accumulate CIA in vacuoles, which is consistent with the mode of action of the other systems. Overall, my assessment of the paper is that the authors may have a nice story, but the transition to plants needs to be better described and potentially supported by additional experiments. For example, the authors seem to conclude that this resistance mechanism will be a very broad spectrum. Is there a second toxin-producing pathogen that could be used to assess whether this is true?

   Thanks for your advice! To answer the question that "Is there a second toxin-producing pathogen that could be used to assess whether this is true?", we added the data of another t toxin-producing pathogens, Fusarium oxysporum and another V. dahliae race, L2-1, to support the conclusion that BbCrpa can confer resistance of plants against pathogens. As expected, the expression of BbCRPA in Arabidopsis and cotton could also significantly increase the resistance to the pathogens we tested. New data were shown in Figure 5-figure supplement 1G-J.

   Reviewer #2 (Public Review):

   The fungus B. bassiana is one of few fungal species resistant to cyclosporine A and tacrolimus, naturally occurring microbial compounds with antifungal and immunosuppressive properties. The authors studied the mechanism of this resistance and found a novel vesicle-mediated transport pathway that directs the compounds to vacuoles for degradation. This hitherto unknown mode of detoxification is initiated by the activity of a phospholipid flippase of the P4-ATPase type. Interestingly, transgenically expressing the fungal flippase in plant model systems induces a similar detoxification pathway and makes the plants resistant to certain fungal toxins of secondary metabolism.

   Strengths

   The genetic screening, isolation of cyclosporine A (CsA) resistant mutants, and characterization of the causative gene BbCrpa are very solid with two independent alleles, a synthetic knockout strain, and rescue of the mutant phenotype.

   BbCrpa protein function in detoxification is demonstrated convincingly by expression in another CsA-sensitive strain, as is its reliance on sites conveying ATPase activity for proper function. It is also functionally different from a relatively closely related P4-ATPase from yeast.

   Using fluorescently labeled CsA and tacrolimus (FK506), it is nicely demonstrated how the compounds are going through the anterograde pathway all the way to the vacuole.

   The authors demonstrate that vacuolar targeting is key for the detoxifying function of BbCrpa and identify the targeting motif that contains a ubiquitination site.

   A trans-species approach (actually, trans-kingdom) confers that BbCrpa can also enhance vacuolar targeting of small toxic compounds to vacuoles in plants, which is quite astounding, given that plant endomembrane transport has quite a number of differences from that of fungi.

   Weaknesses

   It is not clear at which temporal scale CsA is going through the different endosomal compartments.

   Thanks for your comments! We agree your idea that it is better to provide indication of the temporal scale of CsA entering into different endosomal compartments. Actually, we had tried to trace the distribution of CsA in cells many times. Unfortunately, the fluorescence of 5-FAM is weak and decreases fast compared with eGFP or mRFP protein, which made us difficult to capture the transient localization and moving trace of CsA in the cells. Nevertheless, the trail of BbCrpa, which carried CsA from the vesicles to early/late endosome, and vacuoles, can reflect the pathway of the cargo (Figure 3K, Supplementary file 1).

   Can it be ruled out that the fluorescently labeled CsA and the GFP-tagged BbCrpa are stripped off their label and we are seeing the free label only?

   Thanks! We accept your comments. In order to rule out the interference from the cleaved fluorescent proteins (i.e., eGFP and mRFP) or chemical compound (i.e. 5-FAM), we took eGFP/mRFP and 5FAM as control. New data about the localization of eGFP/mRFP and 5-FAM in B. bassiana hypha were provided in the revised manuscript. Our observation indicated that the distribution of fluorescent materials alone is different with the labeled ones, confirming the bona fide localization of the fusion proteins or compound. Please see Figure 2-figure supplement 2.

   Reviewer #3 (Public Review):

   In this manuscript, the authors have attempted to determine the molecular mechanisms underlying the resistance of an insect fungal pathogen Bauveria barbicans to cyclosporine A (CsA) and tacrolimus (FK506), known antifungal secondary metabolites that are also used extensively as immunosuppressing agents in medicine. By screening the random insertion mutant library of this pathogen, they identified the gene responsible for conferring resistance to CsA and FK506. The amino acid sequence of the gene identified it to be P4-ATPase, designated BbCrpa, which was hypothesized to be involved in vesicle-mediated transport. The identity of this gene as a CsA resistance gene was confirmed by demonstrating that disruption of this gene in B. barbicans confers susceptibility to CsA and FK506 and the expression of the wild-type gene in the BbCRPA knockout strain restores resistance to these compounds. In addition, expression of this gene in a plant pathogen Verticillium dahliae confers resistance to CsA and FK506.

   The authors hypothesized that CsA/FK506 detoxification in the resistant B. barbiana strain used is through the BbCRPA-mediated vesicle transport process transporting these toxic metabolites to vacuoles through trans-Golgi (TGN)-early endosome (EE)-late endosomes (LE) pathway. To test this hypothesis, they employed a dual labeling system using 5-carboxyfluorescein fluorescently labeled CsA and FK506 and fusions of red fluorescent proteins (RFP) with BbRab5 GTPase (a marker for early endosomes), BbRab7 GTPase (a marker for late endosome) and pleckstrin homology domain of human oxysterol binding protein (PHOSBP) (a marker for trans-Golgi). By looking at the distribution of fluorescein-labeled CsA and FK506 in the wild-type and ΔBbCRPA cells using confocal microscopy, the authors have provided compelling evidence that these metabolites are transported to the vacuole. The co-localization of CsA with endocytic marker proteins also appears to be convincing for the most part. The co-localization of CsA with mRFP:: PHOSBP as shown in Fig. 2D seems less compelling. Also, in the confocal micrographs presented in Fig. 2, the distinction between early and late endosomes seems less convincing. It seems that there is significant heterogeneity in the early endosome and late endosome populations in the fungal cells.

   1. The co-localization of CsA with mRFP::PHOSBP as shown in Fig. 2D seems less compelling.

   Thanks a lot! According to your suggestion, we repeated the observation and replaced the original figure with new one (Figure 2D). The new figures clear indicates the co-localization of CsA with mRFP::PHOSBP.

   1. Also, in the confocal micrographs presented in Fig. 2, the distinction between early and late endosomes seems less convincing. It seems that there is significant heterogeneity in the early endosome and late endosome populations in the fungal cells.

   Thanks! We agree with your comments! Rab5 is widely used as a marker for early endosomes, while Rab7 is used as a marker for late endosomes. Nevertheless, early endosome and late endosome are hardly to be distinguished strictly. According to your suggestion, we repeated the observation, and replaced the Figure 2F and 2L, and Figure 3E with new ones. Our results indicated that mRFP::BbRab5 appeared largely in the lumen of vacuoles and some punctaes (early endosomes), and mRFP::BbRab7 locates to vacuolar membrane and late endosomal compartments. These can be seen in our observations in Figure 3D and 3E, which are consistent with the observations in Fusarium graminearum described by Zheng et al. (Zheng et al., 2018, New Phytologist, 219: 654671, DOI: 10.1111/nph.15178).

   The authors addressed the question of whether BbCrpa acts as a component involved in vesicle trafficking through the trans-Golgi-endosomes to vacuoles. Ten different eGFP-BbCrpa fusion proteins were constructed and shown to provide detoxification of CsA and FK506. The BbCrpa is localized to the apical plasma membrane and spitzenkorper region of the germ tube. The evidence for localization of BbCrpa in trans-Golgi and vacuole is clear. However, the experimental data shown in Fig. 3D-F claiming localization of BbCrpa in EEs and LEs are somewhat difficult for this reviewer to interpret. It is also not clear to this reviewer why the two FM4-64 staining patterns in Fig. 3C and Fig. 3F are strikingly different. The evidence for co-localization of the fluorescein-labeled CsA or FK506 with RFP-labeled BbCrpa in vacuoles (Fig.3 H and J) is convincing. Figs. 3L-M depicting dynamic trafficking of BbCrpa from TGN to vacuoles using timelapse microscopy is interesting. In Fig. 3M, eGFP should be labeled eGFP::Drs2p. The authors have identified the N-terminal vacuole targeting motif in BbCrpa and shown that the C-terminal sequence from aa1326 to aa1359 is important for detoxification of CsA and FK506 in B. barbiana. In particular, the importance of three Tyr residues located in the C-terminal domain of the enzyme for CsA resistance is interesting.

   1. The experimental data shown in Fig. 3D-F claiming localization of BbCrpa in EEs and LEs are somewhat difficult for this reviewer to interpret.

   Thanks for your comments! P4-ATPases are implicated in the initiation of vesicle biogenesis and moves along with the vesicle (Panatala et al., 2015, Journal of Cell Science, 128: 2021-2032, DOI: 10.1242/jcs.102715; van der Mark et a., 2013, International Journal of Molecular Sciences, 14, 7897-7922, DOI: 10.3390/ijms14047897). In this study, the crucial issue to be addressed was the journey of CsA to the vacuole, which might be through BbCrpa-mediated TGN-EE-LE vesicle transport pathway. Therefore, we observed the localization of BbCrpa in TGN, EEs and LEs. It has been reported that small GTPase Rab5 is localized to the early endosomes (Bucci et al., 1992, Cell, 70: 715-728, DOI: 10.1016/0092-8674(92)90306-w), while Rab7 is to the late endosomal compartment (Vitelli et al., 1997, The Journal of Biological Chemistry, 272: 4391-4397, DOI: 10.1074/jbc.272.7.4391). Thus, Rab5 and Rab7 are used as marker proteins to indicate EEs and LEs, respectively. Nevertheless, Rab5 and Rab7 could also be observed in MVB (multivesicular bodies) or vacuoles (Toshima J.Y.,et al., 2014, Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole, Nature Communication, 5:3498, DOI: 10.1038/ncomms4498; Zheng et al., 2018, New Phytologist, 219: 654-671, DOI: 10.1111/nph.15178). According to your suggestion, we repeated our observation and replaced Figure 3E with new one. In Figure3D, we can see mRFP::BbRab5 appeases largely in the lumen of vacuoles and some punctaes (early endosomes), and in Figure 3E mRFP::BbRab7 locates to late endosomal compartments and vacuolar membrane, which are consistent with the observations in Fusarium graminearum described by Zheng et al.(Zheng et al., 2018, New Phytologist, 219: 654671, DOI: 10.1111/nph.15178).

   1. It is also not clear to this reviewer why the two FM4-64 staining patterns in Fig. 3C and Fig. 3F are strikingly different.

   Thanks for your comments! FM4-64 is a styryl dye that can bind to the outer lipid leaflet of the plasma membrane and enter into cells through endocytosis (Scheuring et al., 2015, Methods in Molecular Biology, 1242:83-92, DOI: 10.1007/978-1-4939-1902-4_8). When the dye is internalized, it can be observed firstly in the membrane of vesicles and endosomal compartments, and then appears in the vacuolar membrane (Jelníková et al., 2010, Plant Journal, 61(5): 883-892, DOI: 10.1111/j.1365-313X.2009.04102.x; Löfke et al., 2013, Journal of Integrative Plant Biology, 55(9): 864-875, DOI: 10.1111/jipb.12097). In Figure 3C, we tried to show the evidence that eGFP::BbCrpa appears in vesicles that are stained by FM4-64, while in Figure 3F, we aimed to indicate eGFP::BbCrpa accumulates in mature vacuoles. Hence, FM4-64 staining patterns in Figure 3C and Figure 3F are somehow different.

   1. In Fig. 3M, eGFP should be labeled eGFP::Drs2p.

   Thanks for your reminder! We have modified it according to the suggestion. Please see Figure 3M.

   Finally, the authors overexpressed BbCrpa gene in transgenic Arabidopsis and cotton plants to show that transgenic plants expressing this enzyme are protected from the toxic effects of the toxin cinnamyl acetate (CA) produced by the fungal pathogen Verticillium dahlia which causes vascular wilt disease in these plants. The data reported in Fig. 5A show that the transgenic Arabidopsis seed is able to germinate in presence of CA, whereas the nontransgenic control seed is not able to germinate. Evidence is presented that CA accumulates in the vacuole in transgenic Arabidopsis. However, the seedlings emerging from transgenic seeds are only partially protected from CA (Fig. 5A). It is also clear from the data presented in Figs. 5B-G that expression of the BbCrpa gene in transgenic Arabidopsis and cotton affords protection from infection by V. dahlia although no evidence for the expression of this gene at the protein level is presented. However, it seems likely that the transgenic lines only show delayed disease symptoms and are not truly resistant to this pathogen. The authors did not state clearly if Verticillium wilt disease resistance assays were performed on homozygous transgenic plants and their corresponding null segregants as negative controls. They also fail to provide evidence that the transgenic Arabidopsis and cotton challenged with the pathogen are able to grow to maturity and set viable seeds.

   1）However, the seedlings emerging from transgenic seeds are only partially protected from CA (Fig. 5A).

   Thanks for your comments! In this study, at the concentration of 50 μg/ml, the germination of wildtype seeds of Arabidopsis was severely inhibited while the transgenic seeds were still able to germinate. However, the growth of transgenic seedling was suppressed obviously compared with that of the untreated seedlings (Figure 5A). According to your suggestion, in the revised manuscript, we used “tolerance”, rather than “resistance” to weaken the statement.

   1. However, it seems likely that the transgenic lines only show delayed disease symptoms and are not truly resistant to this pathogen. The authors did not state clearly if Verticillium wilt disease resistance assays were performed on homozygous transgenic plants and their corresponding null segregants as negative controls. They also fail to provide evidence that the transgenic Arabidopsis and cotton challenged with the pathogen are able to grow to maturity and set viable seeds.

   Thanks for your comments! In our routine procedure for generating transgenic plant lines, we identified non-transgenic plants in the segregative generation (usually in T1 generation) of transformats, and then used these non-transgenic plants (null lines) as control to rule out the somatic variation from tissue culture. In the meantime, the homologous transgenic plants were identified in the segregative generation and propagated by selfing. Relevant descriptions were added in the section of Methods & Materials (Lines 783-795).

   We agree with your opinion. The resistance displayed in seedlings does not always match that in maturity stage, because the resistance of host to Verticilium pathogen can be affected by environmental conditions, for example, temperature and nutrition. Nevertheless, in the case of our transgenic plants, the resistance to Verticilium disease is endowed from the detoxic function of BbCrpa. Theoretically, such transgenic traits will not be significantly affected by the environmental conditions if the expression of transgenes is stable. We had detected the expression level of BbCRPA during plants growth and in deferent generation (to T5 generation). The expression of BbCRPA gene was stable and the resistance to the diseases is descendible.

   Read the original source
3. 

   eLife

   Jul 13, 2022

   Evaluation Summary:

   In this manuscript, the authors focus on the fungus B. bassiana, which is resistant to the toxin cyclosporine A. Through a mutant screen, the authors identify the key gene that mediates the sequestration of the toxin in vacuoles. They further show that this gene can be transferred to a distinct fungus and also to plants to protect against a toxin-producing fungal pathogen. Therefore, this work may lead to novel disease control strategies against fungal pathogens.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
4. 

   eLife

   Jul 13, 2022

   Reviewer #1 (Public Review):

   This manuscript describes experiments that lead to a potentially impactful result and most of the data seem very nice. The authors conducted a mutant screen to find the gene BbCrpa from a fungus resistant to cyclosporine A (CsA). Microscopy indicates that the mode of action is likely sequestration of the toxin in vacuoles, mediated through the P4-ATPase pathway. They also show that expression of BbCrpa in Verticillium renders that fungus resistant to CsA. The paper then takes a very large jump across kingdoms and toxins and asks if BbCrpa, expressed in plants, will confer resistance to a different toxin (cinnamon acetate) that is produced by Verticillium. They conduct disease assays on Arabidopsis and cotton and show promising results, but these assays are less thoroughly completed. They provide microscopic …

   Reviewer #1 (Public Review):

   This manuscript describes experiments that lead to a potentially impactful result and most of the data seem very nice. The authors conducted a mutant screen to find the gene BbCrpa from a fungus resistant to cyclosporine A (CsA). Microscopy indicates that the mode of action is likely sequestration of the toxin in vacuoles, mediated through the P4-ATPase pathway. They also show that expression of BbCrpa in Verticillium renders that fungus resistant to CsA. The paper then takes a very large jump across kingdoms and toxins and asks if BbCrpa, expressed in plants, will confer resistance to a different toxin (cinnamon acetate) that is produced by Verticillium. They conduct disease assays on Arabidopsis and cotton and show promising results, but these assays are less thoroughly completed. They provide microscopic evidence that the transgenics accumulate CIA in vacuoles, which is consistent with the mode of action of the other systems. Overall, my assessment of the paper is that the authors may have a nice story, but the transition to plants needs to be better described and potentially supported by additional experiments. For example, the authors seem to conclude that this resistance mechanism will be a very broad spectrum. Is there a second toxin-producing pathogen that could be used to assess whether this is true?

   Read the original source
5. 

   eLife

   Jul 13, 2022

   Reviewer #2 (Public Review):

   The fungus B. bassiana is one of few fungal species resistant to cyclosporine A and tacrolimus, naturally occurring microbial compounds with antifungal and immunosuppressive properties. The authors studied the mechanism of this resistance and found a novel vesicle-mediated transport pathway that directs the compounds to vacuoles for degradation. This hitherto unknown mode of detoxification is initiated by the activity of a phospholipid flippase of the P4-ATPase type. Interestingly, transgenically expressing the fungal flippase in plant model systems induces a similar detoxification pathway and makes the plants resistant to certain fungal toxins of secondary metabolism.

   Strengths:

   The genetic screening, isolation of cyclosporine A (CsA) resistant mutants, and characterization of the causative gene BbCrpa are …

   Reviewer #2 (Public Review):

   The fungus B. bassiana is one of few fungal species resistant to cyclosporine A and tacrolimus, naturally occurring microbial compounds with antifungal and immunosuppressive properties. The authors studied the mechanism of this resistance and found a novel vesicle-mediated transport pathway that directs the compounds to vacuoles for degradation. This hitherto unknown mode of detoxification is initiated by the activity of a phospholipid flippase of the P4-ATPase type. Interestingly, transgenically expressing the fungal flippase in plant model systems induces a similar detoxification pathway and makes the plants resistant to certain fungal toxins of secondary metabolism.

   Strengths:

   The genetic screening, isolation of cyclosporine A (CsA) resistant mutants, and characterization of the causative gene BbCrpa are very solid with two independent alleles, a synthetic knockout strain, and rescue of the mutant phenotype.

   BbCrpa protein function in detoxification is demonstrated convincingly by expression in another CsA-sensitive strain, as is its reliance on sites conveying ATPase activity for proper function. It is also functionally different from a relatively closely related P4-ATPase from yeast.

   Using fluorescently labeled CsA and tacrolimus (FK506), it is nicely demonstrated how the compounds are going through the anterograde pathway all the way to the vacuole.

   The authors demonstrate that vacuolar targeting is key for the detoxifying function of BbCrpa and identify the targeting motif that contains a ubiquitination site.

   A trans-species approach (actually, trans-kingdom) confers that BbCrpa can also enhance vacuolar targeting of small toxic compounds to vacuoles in plants, which is quite astounding, given that plant endomembrane transport has quite a number of differences from that of fungi.

   Weaknesses:

   It is not clear at which temporal scale CsA is going through the different endosomal compartments.

   Can it be ruled out that the fluorescently labeled CsA and the GFP-tagged BbCrpa are stripped off their label and we are seeing the free label only?

   Read the original source
6. 

   eLife

   Jul 13, 2022

   Reviewer #3 (Public Review):

   In this manuscript, the authors have attempted to determine the molecular mechanisms underlying the resistance of an insect fungal pathogen Bauveria barbicans to cyclosporine A (CsA) and tacrolimus (FK506), known antifungal secondary metabolites that are also used extensively as immunosuppressing agents in medicine. By screening the random insertion mutant library of this pathogen, they identified the gene responsible for conferring resistance to CsA and FK506. The amino acid sequence of the gene identified it to be P4-ATPase, designated BbCrpa, which was hypothesized to be involved in vesicle-mediated transport. The identity of this gene as a CsA resistance gene was confirmed by demonstrating that disruption of this gene in B. barbicans confers susceptibility to CsA and FK506 and the expression of the …

   Reviewer #3 (Public Review):

   In this manuscript, the authors have attempted to determine the molecular mechanisms underlying the resistance of an insect fungal pathogen Bauveria barbicans to cyclosporine A (CsA) and tacrolimus (FK506), known antifungal secondary metabolites that are also used extensively as immunosuppressing agents in medicine. By screening the random insertion mutant library of this pathogen, they identified the gene responsible for conferring resistance to CsA and FK506. The amino acid sequence of the gene identified it to be P4-ATPase, designated BbCrpa, which was hypothesized to be involved in vesicle-mediated transport. The identity of this gene as a CsA resistance gene was confirmed by demonstrating that disruption of this gene in B. barbicans confers susceptibility to CsA and FK506 and the expression of the wild-type gene in the BbCRPA knockout strain restores resistance to these compounds. In addition, expression of this gene in a plant pathogen Verticillium dahliae confers resistance to CsA and FK506.

   The authors hypothesized that CsA/FK506 detoxification in the resistant B. barbiana strain used is through the BbCRPA-mediated vesicle transport process transporting these toxic metabolites to vacuoles through trans-Golgi (TGN)-early endosome (EE)-late endosomes (LE) pathway. To test this hypothesis, they employed a dual labeling system using 5-carboxyfluorescein fluorescently labeled CsA and FK506 and fusions of red fluorescent proteins (RFP) with BbRab5 GTPase (a marker for early endosomes), BbRab7 GTPase (a marker for late endosome) and pleckstrin homology domain of human oxysterol binding protein (PHOSBP) (a marker for trans-Golgi). By looking at the distribution of fluorescein-labeled CsA and FK506 in the wild-type and ΔBbCRPA cells using confocal microscopy, the authors have provided compelling evidence that these metabolites are transported to the vacuole. The co-localization of CsA with endocytic marker proteins also appears to be convincing for the most part. The co-localization of CsA with mRFP:: PHOSBP as shown in Fig. 2D seems less compelling. Also, in the confocal micrographs presented in Fig. 2, the distinction between early and late endosomes seems less convincing. It seems that there is significant heterogeneity in the early endosome and late endosome populations in the fungal cells.

   The authors addressed the question of whether BbCrpa acts as a component involved in vesicle trafficking through the trans-Golgi-endosomes to vacuoles. Ten different eGFP-BbCrpa fusion proteins were constructed and shown to provide detoxification of CsA and FK506. The BbCrpa is localized to the apical plasma membrane and spitzenkorper region of the germ tube. The evidence for localization of BbCrpa in trans-Golgi and vacuole is clear. However, the experimental data shown in Fig. 3D-F claiming localization of BbCrpa in EEs and LEs are somewhat difficult for this reviewer to interpret. It is also not clear to this reviewer why the two FM4-64 staining patterns in Fig. 3C and Fig. 3F are strikingly different. The evidence for co-localization of the fluorescein-labeled CsA or FK506 with RFP-labeled BbCrpa in vacuoles (Fig.3 H and J) is convincing. Figs. 3L-M depicting dynamic trafficking of BbCrpa from TGN to vacuoles using time-lapse microscopy is interesting. In Fig. 3M, eGFP should be labeled eGFP::Drs2p. The authors have identified the N-terminal vacuole targeting motif in BbCrpa and shown that the C-terminal sequence from aa1326 to aa1359 is important for detoxification of CsA and FK506 in B. barbiana. In particular, the importance of three Tyr residues located in the C-terminal domain of the enzyme for CsA resistance is interesting.

   Finally, the authors overexpressed BbCrpa gene in transgenic Arabidopsis and cotton plants to show that transgenic plants expressing this enzyme are protected from the toxic effects of the toxin cinnamyl acetate (CA) produced by the fungal pathogen Verticillium dahlia which causes vascular wilt disease in these plants. The data reported in Fig. 5A show that the transgenic Arabidopsis seed is able to germinate in presence of CA, whereas the nontransgenic control seed is not able to germinate. Evidence is presented that CA accumulates in the vacuole in transgenic Arabidopsis. However, the seedlings emerging from transgenic seeds are only partially protected from CA (Fig. 5A). It is also clear from the data presented in Figs. 5B-G that expression of the BbCrpa gene in transgenic Arabidopsis and cotton affords protection from infection by V. dahlia although no evidence for the expression of this gene at the protein level is presented. However, it seems likely that the transgenic lines only show delayed disease symptoms and are not truly resistant to this pathogen. The authors did not state clearly if Verticillium wilt disease resistance assays were performed on homozygous transgenic plants and their corresponding null segregants as negative controls. They also fail to provide evidence that the transgenic Arabidopsis and cotton challenged with the pathogen are able to grow to maturity and set viable seeds.

   Read the original source
7. 

   Version published to 10.1101/2022.05.03.490477v1 on bioRxiv

   May 3, 2022