A Eucalyptus Pht1 Family Gene EgPT8 Is Essential for Arbuscule Elongation of Rhizophagus irregularis

ABSTRACT The majority of vascular flowering plants can establish arbuscular mycorrhizal (AM) symbiosis with AM fungi. These associations contribute to plant health and plant growth against various environmental stresses. In the mutualistic endosymbiosis, the AM fungi deliver phosphate (Pi) to the host root through highly branched hyphae called arbuscules. The molecular mechanisms of Pi transfer from AM fungi to the plant have been determined, which are dominated by AM-specific Pi transporters belonging to the PHOSPHATE TRANSPORTER 1 (Pht1) family within the subfamily I. However, it is unknown whether Pht1 family proteins are involved in other regulations in AM symbiosis. Here, we report that the expression of EgPT8 is specifically activated by AM fungus Rhizophagus irregularis and is localized in root cortical cells containing arbuscules. Interestingly, knockdown of EgPT8 function does not affect the Eucalyptus grandis growth, total phosphorous (P) concentration, and arbuscule formation; however, the size of mature arbuscules was significantly suppressed in the RNAi-EgPT8 lines. Heterogeneous expression of EgPT4, EgPT5, and EgPT8 in the Medicago truncatula mutant mtpt4-2 indicates that EgPT4 and EgPT5 can fully complement the defects of mutant mtpt4-2 in mycorrhizal Pi uptake and arbuscule formation, while EgPT8 cannot complement the defective AM phenotype of the mtpt4-2 mutant. Based on our results, we propose that the AM fungi-specific subfamily I transporter EgPT8 has novel functions and is essential to arbuscule elongation. IMPORTANCE Arbuscular mycorrhizal (AM) formation in host root cortical cells is initiated by exchanges of diffusible molecules, among which Pi uptake is known as the important feature of AM fungi on symbiosis functioning. Over the last two decades, it has been repeatedly proven that most vascular plants harbor two or more AM-specific Pht1 proteins; however, there is no direct evidence regarding the potential link among these Pi transporters at the symbiotic interface. This work revealed a novel function of a structurally conserved protein involved in lateral arbuscule development. In total, we confirmed that three AM-specific Pht1 family proteins are nonredundant in Eucalyptus grandis and that EgPT8 is responsible for fungal arbuscule elongation of Rhizophagus irregularis.

and its localization strongly resembles that of previously characterized AM PTs, EgPT8 does not appear to have a function in symbiotic Pi uptake via arbuscules. While the paper does not solve the question of how PHT1:4 and PHT1;8 differ, it shows that they have different roles during AM symbiosis. This paper is interesting and opens multiple interesting lines of research. However, some data representations/interpretations are a bit confusing. Please see below for details.
Title: consider deleting the word 'fungal' Line 139/140: the sentence "to examine the sensitivity of EgPT8 expression to AM roots of E. grandis" is a bit confusing. Shouldn't it say "to determine the expression level of EgPT8 in E. grandis roots at different time points of AM symbiosis"?
Line 153 ff: The authors state in the text that GFP-EgPT8 co-localizes with ER maker in N. benthamiana leaves. Looking at Fig  S4, I am not so sure this conclusion is necessarily biologically relevant. To me it seems like there may also be at least partial colocalization with the plasma membrane marker. In our experience, N. benthamiana over-expression often results in ER accumulation of fusion proteins, but this may simply be due to very high gene expression. I am not sure we can conlude that "EgPT8 were resident in the ER of N. benthamiana leaf epidermal cells in nonmycorrhizal tissue" (line 155/156). The fluorescence image in Fig 2 is more informative since it uses the endogenous EgPT8 promoter and the tissue in which the protein normally is expressed (roots). However, although these images look very much like the PAM-localization that has been previously shown for e.g. Medicago PT4 and other PTs, I feel the sentence "This evidence indicates that EgPT8 is exclusively localized to the PAM" (line 163/164 and also 170) is not warranted as co-localization of EgPT8 to the PAM was not shown (and sub-cellular localization was concluded to be in the ER). This experiment has to be performed or the sentence and all PAMrelated sentences throughout the whole manuscript need to be tuned down.
Line 189 ff: The sentence "the growth rate of mutant EY917 carrying the pFL61-EgPT8 is significantly higher than that of EY917 cells with pFL61 at the logarithmic phase (from 8 to 20 h; Fig. 3D), however, is lower than EgPT4 and EgPT5 expressing in EY917" does not make sense with what is shown in Figure 3D. If I interpret the figure correctly, PT4 is higher but PT5 is lower than PT8? Also, there are no statistical tests shown in Fig. 3 or the text, so the word "significantly" is not justified.
Line 224: AsPT1 is not involved in symbiotic Pi uptake     Figure 3E: What do the significance asterisks refer to? According to the figure legend, a Tukey test was performed but it is unclear which of the pairwise comparisons is shown. This is by the way the case for many of the main and supplementary figures. This test is only applicable when comparing 2 samples. When more than 2 samples are being analyzed, an anove test should be performed. Which sample was the control for the t-test? Figure 6A g-l: It would be very helpful to scale these images to the same size. The way it is represented now, it is impossible for the reader to inspect the arbuscule sizes. Reviewer #2 (Comments for the Author): The submitted paper by Che and colleagues considers the role of the PT1;8 family of putative phosphate transporters, with a special focus on EgPT8. They undertake a range of in silico and experimental approaches to advance our understanding of this gene during AM symbioses. Their research models are varied and appropriate for the work being undertaken. The paper is generally well written with solid results and a nice interpretation of the work without over-extending the conclusions. This paper contributes nicely to the field. I only have a few minor editorial comments: Line 53: "Symbionts" should be "symbioses" Line 105-106: you need to reference this "previous" work Line 221: This is a complicated sentence due to the double negative. It would be clearer just to say that "EgPT8 is dispensable...." Line 225, 230: remove "as expected" Line 286: "eucalypts" should be "ecualypt" Line 297: "arbuscule" should be plural Line 331: "Token" should be "taken" Staff Comments:

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Dear Editor and Reviewers,
We would like to thank you for your careful reading, helpful comments, and constructive suggestions, which has significantly improved the presentation of our manuscript. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in MS Word. We have carefully considered all comments from the reviewers and revised our manuscript accordingly. In the following section, we summarize our responses to each comment from the reviewers. The comments are reproduced and our responses are given directly afterward. We explain the appropriateness of statistical tests and how statistics are reported. We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. We believe that our responses have well addressed all concerns from the reviewers. We hope our revised manuscript can be accepted for publication. We hope that the revised manuscript is accepted for publication in the Microbiology Spectrum.

Comment 1: Title: consider deleting the word 'fungal'
Reply 1: Thanks for the reviewer's kind suggestion. We have deleted the word 'fungal' from the title (Please see Line 1).

The fluorescence image in Fig 2 is more informative since it uses the endogenous
EgPT8 promoter and the tissue in which the protein normally is expressed (roots).
However, although these images look very much like the PAM-localization that has been previously shown for e.g. Medicago PT4 and other PTs, I feel the sentence "This evidence indicates that EgPT8 is exclusively localized to the PAM" (line 163/164 and also 170) is not warranted as co-localization of EgPT8 to the PAM was not shown (and sub-cellular localization was concluded to be in the ER). This experiment has to be performed or the sentence and all PAM-related sentences throughout the whole manuscript need to be tuned down.
Reply 3: Thank you for your comment. Our result of membrane localization of EgPT8 is consistent with previous report that the AM-specific proteins is able to polarly localize to the PAM upon AM symbiosis to facilitate the symbiotic Pi uptake (Pumplina et al., 2012). However, in the tobacco leaves or other plant tissues where cannot form the AM symbiotic system, resulting in the polar targeting of EgPT8.
Once arbuscules are formed in the tissue, the AM-specific proteins will be recruited to the correct location (Che et al., 2022). Further, the full-length sequences of 17 of the 22 Pi transporters of E. grandis were isolated from different tissues, in which the relative expression levels of two genes were higher in root tissues than other E. grandis PHT1 genes including EgPT4, EgPT5 and EgPT8. However, the two genes were all co-localized with the plasma membrane marker (The data have not been published).
Moreover, we have also presented subcellular localization of the EgPT8 transporter in yeast cells, out result demonstrated that EgPT8 protein is targeted to the plasma membrane of yeast cells (Fig. 3C). According your advices, all PAM-related sentences throughout the whole manuscript have been revised. We hope that our data is sufficient to give the conclusion that EgPT8 is localized in AM fungi-colonized cortical cells.   Fig. 3D), however, is lower than EgPT4 and EgPT5 expressing in EY917" does not make sense with what is shown in Figure 3D. If I interpret the figure correctly, PT4 is higher but PT5 is lower than PT8?
Also, there are no statistical tests shown in Fig. 3 or the text, so the word "significantly" is not justified.

Reply 4:
We appreciate for your valuable comment. We have revised the text to address your concerns and hope that it is now clearer. Please see Lines 188-192 of the revised manuscript. The revised details are as follows: Next, we introduced the pFL61-EgPT8 into EY917 to text the growth tendency. In agreement with the complementation of EgPT8 in EY917, the growth rate of mutant EY917 carrying the pFL61-EgPT8 is higher than that of EY917 cells with pFL61 or pFL61-EgPT5 at the logarithmic phase (from 8 to 20 h; Fig. 3D), however, is lower than EgPT4 expressing in EY917. When the EgPT8-RNAi lines were harvested at 49 dpi under low Pi conditions, we identified three lines by qRT-PCR determination, EgPT8-RNAi-1, EgPT8-RNAi-2, and EgPT8-RNAi-3, showing significantly decreased expression levels of EgPT8 (Fig.   4B).

Comment 6: Line 224: AsPT1 is not involved in symbiotic Pi uptake
Reply 6: Thank you for your significant reminding. An earlier study has determined that AsPT1 is required for AM symbiosis, however, AsPT1 is not involved in symbiotic Pi uptake. To demonstrate whether EgPT8 is indispensable for arbuscule formation, we qualified the mycorrhization in the RNAi-EgPT8 lines and control lines (Please see lines 223-225). Figure 1: it would be helpful for the reader to highlight the eucalyptus PTs discussed in this manuscript.

Comment 7:
Reply 7: We appreciate to you for your valuable advice. The eucalyptus PTs in Figure