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  1. Author Response

    Reviewer #1 (Public Review):

    1. Fig 6E shows that CAPE1 is released only upon Fol infection. This appears to contradict with the notion that FolSpv1 prevents CAPE1 release. However, Fol strain overexpressing FolSpv1 prevented the release of CAPE1. It is necessary to compare WT and the mutant strain in which the FolSvp1 gene is deleted. One would expect that the mutant strain induces significantly more CAPE1 release. Similarly, mutant strain complemented with the nls1 construct needs to be tested to see whether nuclear localization is required for preventing CAPE1 release.

    Thank you for the good suggestions! According to the revision policy of eLife in response to COVID-19, we stated in the Discussion section that FolSpv1-mediated translocation of SlPR1 into the nucleus impedes CAPE1 release needs to be further strengthened with additional data in the revised manuscript (lines 441-444). We would like to perform the suggested experiments in future studies.

    1. SlPR1 is localized in the apoplast in a manner dependent on the signal peptide (Fig 5-figure supplement 1). Overexpression of SlPR1 with added NLS but lacking the signal peptide failed to enhance disease resistance to Fol infection (Fig 7G). What about overexpression of SlPR1 lacking the signal peptide without the added NLS? Does retention of SlPR1 in the cytoplasm sufficient to abolish its function? It is not even discussed why SlPR1 has to be in the nucleus to prevent CAPE1 release.

    Thank you for these suggestions! We have discussed the possibility that binding of FolSvp1 to SlPR1 may inhibit the function of the latter in the cytoplasm and stated that additional experiments are required in future studies in the revised manuscript (lines 436-444).

    1. FolSvp1 carrying the PR1 signal peptide interacted with SlPR1 in the apoplast (Fig 6D and Fig 6-figure supplement 2). Why weren't these proteins translocated into the nucleus? These seem to contradict the in vitro uptake data. It seems that either no or only a very small proportion of SlPR1 transiently expressed in tobacco cells is located in the nucleus. Fig 7C shows that infection of the WT strain, but not the nls1 mutant strain, allowed detection of SlPR1 in the nucleus of tomato cells. However, it is not clear how much of SlPR1 remain in the apoplast or cytoplasm. Is the FolSpv1 protein secreted by Fol sufficient to translocate a significant portion of SlPR1 into the nucleus? The authors are suggested to examine apoplastic and cytoplasmic protein fractions for the relative amounts of SlPR1 after Fol infection.

    Thank you very much for this constructive point! The observations of FolSvp1 and SlPR1 interaction in both the apoplast and the nucleus of N. benthamiana leaves suggest that binding of FolSvp1 to SlPR1 may inhibit its anti-fungal activity and/or the cleavage of SlPR1 to produce CAPE1 in the extracellular region or even the cytoplasm. In addition, the BiFC assays performed with N. benthamiana leaves might not completely mimic the physiological conditions. Therefore, whether FolSpv1-mediated translocation of SlPR1 into the nucleus impedes CAPE1 release is the only way of PR1 inactivation needs to be further strengthened with additional data in future studies. We have added these information to the revised manuscript (lines 436-444).

    1. Fig 7J and 7K, a better experiment would be to pretreat WT tomato plants with CAPE1 prior to inoculation with WT and FolSpv1 OE strains. The pretreatment should eliminate the virulence function of FolSpv1 OE if the virulence is solely dependent on the prevention of CAPE1 release.

    Thank you for this suggestion! We have stated in the Discussion section that FolSpv1-mediated translocation of SlPR1 into the nucleus impedes CAPE1 release needs to be further strengthened with additional data in the revised manuscript (lines 441-444). It will be of considerable interest to perform the suggested experiments in future studies.

    Reviewer #2 (Public Review):

    1. As far as I know, the apoplastic PR1 proteins may have a fungicide activity. When the authors tested the interaction between FolSvp1 and SlPR1 in Nicotiana benthamiana by BiFC, both apoplastic and nuclear interactions could be detected. Therefore, the authors should discuss the possibilities whether the binding of FolSvp1 to SlPR1 remained in the apoplast can inhibit (i) its anti-Fol activity and (ii) the cleavage of SlPR1 to produce the CAPE1 peptide. In other words, although translocating SlPR1 to the nucleus by FolSvp1 is effective for suppressing CAPE1 production, this may not be the only way.

    Thank you very much for this constructive point! The observations of FolSvp1 and SlPR1 interaction in both the apoplast and the nucleus of N. benthamiana leaves suggest that binding of FolSvp1 to SlPR1 may inhibit its anti-fungal activity and/or the cleavage of SlPR1 to produce CAPE1 in the extracellular region or even the cytoplasm. Therefore, whether FolSpv1-mediated translocation of SlPR1 into the nucleus impedes CAPE1 release is the only way of PR1 inactivation needs to be further strengthened with additional data in future studies. According to the revision policy of eLife in response to COVID-19, we have added these information to the revised manuscript (lines 436-444).

    1. The FolSvp1 produced in N. benthamiana was using the SlPR1 signal peptide and lacked the acetylation modification. It is possible that the acetylation of FolSvp1 can affect the interaction affinity or localization between FolSvp1 and SlPR1. The K167Q mutation of FolSvp1 might not be able to faithfully mimic the K167 acetylation.

    Thank you for this suggestion! It’s true that the BiFC assays performed with N. benthamiana leaves might not completely mimic the physiological conditions. We have discussed this possibility in the revised manuscript (lines 439-444).

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  2. eLife assessment

    The authors provided strong evidence that the Fusarium oxysporum effector protein FolSpv1 enhances virulence by targeting tomato SlPR1 and preventing the generation of the SlPR1-derived phytocytokine CAPE1, which otherwise positively regulates disease resistance in tomato plants. Strikingly, they show that FolSpv1 translocates SlPR1 from the apoplast back into the nucleus of tomato cell, suggesting a previously unknown mechanism employed by pathogenic microbes.

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  3. Reviewer #1 (Public Review):

    Pathogen effectors promote parasitism either in the apoplast or cytoplasm. Unexpectedly, the work described here suggests that FolSpv1 first interacts with SlPR1 in the apoplast and then translocates SlPR1 into the nucleus of tomato plant cells. The authors suggested that the FolSpv1-mediated translocation of SlPR1 into the nucleus prevented the generation of CAPE1, leading to compromised immunity in tomato plants. The study additionally showed that acetylation of FolSpv1 K167 protects the protein from ubiquitination and proteasome-mediated degradation in both the fungal cell and plant cell. Overexpression of SlPR1 or exogenous application of CAPE1 enhanced resistance to F. oxysporum, indicating that CAPE1 contributes to disease resistance to the pathogen in tomato plants. This is consistent with prior reports that CAPE1 positively regulates plant immunity. Y2H screen followed by BiFC and co-IP supported SlPR1 as a target of FolSpv1. Most importantly, incubation of the SlPR1 recombinant protein with FolSvp1 led to uptake of both FolSvp1 and SlPR1 by tomato root protoplasts and nuclear localization of both proteins. Consistent with their model, NLS sequence is required for FolSpv1 virulence function and re-localization of SlPR1 in the nucleus. Furthermore, disease resistance conferred by SlPR1 overexpression in tomato plants could be reversed by overexpression of FolSpv1 in the fungus. Overall, the work represents a potentially significant advance in effector biology of phytopathogens. However, it is too early to exclude the possibility that the nucleus-dependent virulence function of FolSpv1 is independent of CAPE1. It is a bit strange why nuclear localization of SlPR1 is required for preventing CAPE1 generation. The following concerns need to be addressed.

    1. Fig 6E shows that CAPE1 is released only upon Fol infection. This appears to contradict with the notion that FolSpv1 prevents CAPE1 release. However, Fol strain overexpressing FolSpv1 prevented the release of CAPE1. It is necessary to compare WT and the mutant strain in which the FolSvp1 gene is deleted. One would expect that the mutant strain induces significantly more CAPE1 release. Similarly, mutant strain complemented with the nls1 construct needs to be tested to see whether nuclear localization is required for preventing CAPE1 release.
    2. SlPR1 is localized in the apoplast in a manner dependent on the signal peptide (Fig 5-figure supplement 1). Overexpression of SlPR1 with added NLS but lacking the signal peptide failed to enhance disease resistance to Fol infection (Fig 7G). What about overexpression of SlPR1 lacking the signal peptide without the added NLS? Does retention of SlPR1 in the cytoplasm sufficient to abolish its function? It is not even discussed why SlPR1 has to be in the nucleus to prevent CAPE1 release.
    3. FolSvp1 carrying the PR1 signal peptide interacted with SlPR1 in the apoplast (Fig 6D and Fig 6-figure supplement 2). Why weren't these proteins translocated into the nucleus? These seem to contradict the in vitro uptake data. It seems that either no or only a very small proportion of SlPR1 transiently expressed in tobacco cells is located in the nucleus. Fig 7C shows that infection of the WT strain, but not the nls1 mutant strain, allowed detection of SlPR1 in the nucleus of tomato cells. However, it is not clear how much of SlPR1 remain in the apoplast or cytoplasm. Is the FolSpv1 protein secreted by Fol sufficient to translocate a significant portion of SlPR1 into the nucleus? The authors are suggested to examine apoplastic and cytoplasmic protein fractions for the relative amounts of SlPR1 after Fol infection.
    4. Fig 7J and 7K, a better experiment would be to pretreat WT tomato plants with CAPE1 prior to inoculation with WT and FolSpv1 OE strains. The pretreatment should eliminate the virulence function of FolSpv1 OE if the virulence is solely dependent on the prevention of CAPE1 release.

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  4. Reviewer #2 (Public Review):

    In this work, the authors were trying to prove the model that the fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol) utilizes the acetyltransferase FolArd1 to induce the acetylation of the K167 residue of the effector protein FolSvp1. This acetylation prevents the K152, K258 and K284 ubiquitination-mediated degradation of FolSvp1 in Fol, and meanwhile inhibits the K167 ubiquitination-mediated degradation of FolSvp1 in tomato plants. In the host plants, FolSvp1 interacts specifically with the apoplastic defense protein SlPR1 and translocates it to the nucleus, which suppresses the SlPR1-derived CAPE1 peptide-induced fungal resistance. Overall, the experiments were well designed and the large amount of data justified most of their conclusions. The work sheds novel insight into the virulence mechanisms of fungal effectors by showing that acetylation modification can stabilize a fungal effector, which is able to mis-localize a key defense protein to dampen the host immunity.

    There are two issues that need to be addressed.

    1. As far as I know, the apoplastic PR1 proteins may have a fungicide activity. When the authors tested the interaction between FolSvp1 and SlPR1 in Nicotiana benthamiana by BiFC, both apoplastic and nuclear interactions could be detected. Therefore, the authors should discuss the possibilities whether the binding of FolSvp1 to SlPR1 remained in the apoplast can inhibit (i) its anti-Fol activity and (ii) the cleavage of SlPR1 to produce the CAPE1 peptide. In other words, although translocating SlPR1 to the nucleus by FolSvp1 is effective for suppressing CAPE1 production, this may not be the only way.

    2. The FolSvp1 produced in N. benthamiana was using the SlPR1 signal peptide and lacked the acetylation modification. It is possible that the acetylation of FolSvp1 can affect the interaction affinity or localization between FolSvp1 and SlPR1. The K167Q mutation of FolSvp1 might not be able to faithfully mimic the K167 acetylation.

    Was this evaluation helpful?