Screening of Haustorium Induction Factors of Phelipanche aegyptiaca Pers. Based on Metabolome Analysis of Cucumis melo L. Root Exudates
Round 1
Reviewer 1 Report
Manuscript entitled: “Screening of haustorium induction factors of Phelipanche aegyptiaca Pers. based on metabolome analysis of Cucumis melo L. root exudates” present detailed metabolomics analysis of root exudates of the host plant Cucumis melo which can influence on parasitism of Phelipanche aegyptiaca. The strength of the work is comprehensive analysis of haustorium-inducing factors (HIFs) present in root exudates of susceptible and resistance cultivars. The research presented in this manuscript could be important in context of finding new solutions to fight with parasitic plants from Orobanchaceae family.
The manuscript is well written in terms of English. In my opinion only few sentences could be rephrased for better understanding.
In line 37 please add or convert ¥ to $;
Line 57 sentence about haustorium development could be rephrased and also citation is needed.
Line 62 – many studies … citation
Line 68 – it would be worth to add area of Cucumis melo cultivation in Xinjiang
Line 104 – cultivars obtained in previous research – lack of citation
Line 129 – Hoagland medium - citation or add the composition of the medium to supplementary materials.
Line 157 – for haustorium could be start as a new paragraph.
Line 206 – DP and CE should be explained
Line 441 – please change development degree to size
Line 472; 483 – upper index (10-4)
All Latin names should be written in italics; in-vitro – italics
Introduction
I kindly ask to add more information about life cycle of P. aegyptiaca as well as allelopathy in context of allelochemicals.
Materials and methods
I assume that C. melo cultivars KR1326 (resistant) and K1076 (susceptible) are resistance to P. aegyptiaca. If so, it would be worth to clarify it.
Please add information about GR24 concentration used in experiments. In line 154 we can find GR24 is used as positive control, however in line 175 as negative control – I kindly ask for explanation.
In section 2.3. we can find information, that all solvents for studied chemicals have been tested to check effects on P. aegyptiaca development. It would be worth to add these results to supplementary materials.
Data analysis – providing scripts used for data analysis (R software) will be additional advantage of this manuscript.
Results
I would suggest to upload higher quality pictures of weeds germination and haustorium formation.
Please consider to change bar charts to box plots in figure 1D-F and figure 4. How many measurements have been made in weeds germination and haustorium formation assays?
Please add some information of MRM metabolite detection multi-peak map. It would be worth to add colour legend for Fig 2A and B.
Please provide some information for root exudates sample mixing used as a quality control - Figure 2E (which biological repetition were mixed with which?)
In section – Identification of differential metabolites to resistance and susceptible C. melo root exudates please comment parameters of FC used for filtering data. Shouldn't the values for up-regulation be the same as for down-regulation?
It is very important to change graphs in figure 3 in a way to point out which metabolite changes are caused by resistance and susceptible C. melo root exudates – Fig 3B;C and D.
All seven selected metabolites were down-regulated in Supplementary Table 3. I highly recommended prepare additional experiment to check how up-regulated metabolites will influence on haustorium induction rate. Moreover it will be worth to test how selected metabolites change weed germination.
Discussion
I kindly suggest adding the following parts to the discussion:
Is it known mechanism of resistance to P. aegyptiaca in C. melo cultivars? Could you discussed it in context of metabolomics data obtained in this study?
At which stage of C. melo development P. aegyptiaca is the most dangerous? Shouldn't exudates be collected in that time point to find novel haustorium-inducing factors (HIFs)?
Two studied cultivars differ in plant size - especially in root morphology. Please discuss a little bit more how it could influence on metabolomic data obtain in this study.
Please discuss results from Miure et al., 2022 (Germination Stimulant Activity of Isothiocyanates on Phelipanche spp.) in context of metabolomics data obtained in your study as well as with your transcriptomic data Yao et al., 2016 (Global Transcriptomic Analysis Reveals the Mechanism of Phelipanche aegyptiaca Seed Germination).
It will be very interesting to discuss metabolic changes observed in P. aegyptiaca development compared to differentially changed allochemicals found in root exudates (Nativ et al., 2017 Metabolic Investigation of Phelipanche aegyptiaca Reveals Significant Changes during Developmental Stages and in Its Different Organs).
In future to study deeper role of HIFs during P. aegyptiaca parasitism, I will suggest validate role of found metabolites in A. thaliana - P. aegyptiaca model. It will be easy to find T-DNA mutants with impaired metabolomic pathways that have been described in your paper (for instance: Clarke et al., 2020 - Multiple immunity-related genes control susceptibility of Arabidopsis thaliana to the parasitic weed Phelipanche aegyptiaca).
Moreover described metabolites could be investigated with different parastitic plants from Orobanchaceae family.
In conclusion, the results presented in this work are very interesting and may contribute to the development of new methods of combating parasitic plants. Particularly important to the work is the metabolomics data, which identify new HIFs.
Author Response
Please see below and in the attachment (figures).
Rsponse to Reviewer 1 Comments
Thank you for your attention and valuable comments on this manusprict. We attach great importance to your suggestions and make the following revision and replies:
Point 1: In line 37 please add or convert ¥ to $;
Response 1: In line 37 refers to Chinese literature, which respresents CNY rather than USD, so we believe that ¥ should be used.
Point 2: Line 57 sentence about haustorium development could be rephrased and also citation is needed.
Response 2: The citation 6, 14, 26 have been added in Line 57.
Point 3: Line 62 – many studies … citation
Response 3: The citation 6, 12, 13, 15 have been added in Line 62.
Point 4: Line 68 – it would be worth to add area of Cucumis melo cultivation in Xinjiang
Response 4: The sentence ”In 2022, the planting area of Cucumis melo L. in Xinjiang was 62,6500 hectares, with a total output of 211,6600 tons” have been added in Line 68.
Point 5: Line 104 – cultivars obtained in previous research – lack of citation
Response 5: The relevant content in line 104 refers to our previous research, but this article is under review and has not been published, so it cannot be cited at this time.
Point 6: Line 129 – Hoagland medium - citation or add the composition of the medium to supplementary materials.
Response 6: The composition of Hoagland medium is shown in Figure S5.
Point 7: Line 157 – for haustorium could be start as a new paragraph.
Response 7: The haustorium induction has been start as a new paragraph in line 157.
Point 8: Line 206 – DP and CE should be explained.
Response 8: Declusteringpotential (DP) and collisionenergy (CE) have been added in Line 206.
Point 9: Line 441 – please change development degree to size.
Response 9: We apologize for failing to understand the meaning of this suggestion.
Point 10: Line 472; 483 – upper index (10-4).
Response 10: Ca2+ in L104, 10−4 – 10−6 in L518 have been corrected.
Point 11: All Latin names should be written in italics; in-vitro – italics
Response 11: All Latin names in the L17, 66 , 99, 102, 128, 136, 452, 462 have been italicized.
Introduction
Point 12: I kindly ask to add more information about life cycle of P. aegyptiaca as well as allelopathy in context of allelochemicals.
Response 12: We added a brief life cycle in the second paragraph of the introduction to lead out the haustorium of P. aegyptiaca. The details are as follows: the life cycle of P.aegyptiaca is the same as that of most the holoparasitic Orobanchaceae, including seed phase, autotrophic growth phase and heterotrophic growth phase. During the seed phase, the seeds of P. aegyptiaca lie dormant in the soil, forming a huge seed bank, which germinates in response to allelochemicals such as strigolactones (SLs) secreted by the host roots and then enters the autotrophic growth phase. Subsequently, the radicle of P.aegyptiaca tends to grow towards the host root and form terminal haustorium under the induction of HIFs during the process of approaching the host root. Then, P. aegyptiaca invaded the host by means of the haustorium, established the parasitic relationship and entered the heterotrophic growth phase.
Materials and methods
Point 13: I assume that C. melo cultivars KR1326 (resistant) and K1076 (susceptible) are resistance to P. aegyptiaca. If so, it would be worth to clarify it.
Response 13: The resistant cultivar KR1326 and susceptible cultivar K1076 used in this study are the melon cultivar resistance identification results of pervious study (this article is under review and has not yet been published). The resistance of KR1326 is shown in the growth and development process of P. aegyptiaca,which blocked in the early tubercle state and late unearthed stage, compared with other melon cultivars.
This content will be published in another article, and what appears in this article may cause conflicts of interest, so it will not be detailed in this article.
Point 14: Please add information about GR24 concentration used in experiments
Response 14: We have marked the concentration of GR24 (1×10-7 mol/L) in line 165, this concentration was obtained by testing in advance, and the result is shown in the figure below.
It was observe that seed germination induction rate of GR24 could reach 80% at the concentration of 10-7 mol/L. Meanwhile, there was no significant difference in seed germination induction rate of GR24 between 10-4 and 10-7 mol/L. Therefore, the concentration of GR24 was selected as 10-7 mol/L.
Point 15: In line 154 we can find GR24 is used as positive control, however in line 175 as negative control – I kindly ask for explanation.
Response 15: the effect of root exudates of KR1326 (resistant) and K1076 (susceptible) on seed germination and haustorium induction of P. aegyptiaca was studied in the section 2.3 “Quantification of seed germination and haustorium induction”. This section is divided into two parts.. The test methods and purposes are slightly different.
In line 154, we studied the effect of root exudates on seed germination, GR24 has been widely used in the seed germination of parasite, so GR24 was used as a positive control in this part.
In line 175, we studied the effect of root exudates on haustorium formation, rather than seed germination. Although GR24 has been widely used in parasitic seed germination, it cannot induce haustorium formation (our pervious experiments on seed germination induced by GR24 have proved this conclusion). Therefore, GR24 was used as a negative control in the study of the effect of root exudates on haustorium formation.
Point 16: In section 2.3. we can find information, that all solvents for studied chemicals have been tested to check effects on P. aegyptiaca development. It would be worth to add these results to supplementary materials.
Response 16: The solvents used in this study were selected according to the solubility of the chemicals, mainly included sterilized water and organic solvents. Before the experiment, we had tested the effects of these solvents on the growth of P. aegyptiaca seeds. Obviously,sterilized water has no effect on seed growth , while some high concentration organic solvents have negative impacts on the seed growth, such as low germination rate and blocked growth, as shown in Figure A below. Therefore, we choose a solvent that dissolves both chemicals and water. When preparing the solvent, we first dissolve chemical in a very low amounts of the solvent and then dilute it to the prescribed concentration with sterilized water. It is observed that the development of P. aegyptiaca seeds were normally, as shown in Figure B below, suggesting that this treatment had no significant impact on the growth of P. aegyptiaca.
Point 17: Data analysis – providing scripts used for data analysis (R software) will be additional advantage of this manuscript.
Response 17: The data analysis scripts has been placed in the "Data analysis scripts" file and submitted to the editor at the same time.
Results
Point 18: I would suggest to upload higher quality pictures of weeds germination and haustorium formation. Please consider to change bar charts to box plots in figure 1D-F and figure 4. How many measurements have been made in weeds germination and haustorium formation assays?
Response 18: Figure 1D-F and Figure 4 have been change to box plots, and we have uploaded as clear figure as possible.
In weed germination and haustorium formation assays, 6 biological replicates were set for each treatment and 30 P. aegyptiaca seeds were measured for each replicate. The above assays were carried out for three times.
Point 19: Please add some information of MRM metabolite detection multi-peak map. It would be worth to add colour legend for Fig 2A and B.
Response 19: A color line in the MRM metabolite detection multi-peak map represents one detected metabolite. If color legends are added, there will be messy. Therefore, this information is shown in “Table S2. The information of all identified metabolites is annotated and below the image for readers' understanding.
Point 20: Please provide some information for root exudates sample mixing used as a quality control - Figure 2E (which biological repetition were mixed with which?)
Response 20: Equal volume root exudates samples of KR1326 and K1076 were mixed for quality control (Mix). We mixed all 6 samples (two cultivars, three replicates), and then obtained 3 mix samples to monitor the stability and reliability of the detection process.
Point 21: In section – Identification of differential metabolites to resistance and susceptible C. melo root exudates please comment parameters of FC used for filtering data. Shouldn't the values for up-regulation be the same as for down-regulation?
Response 20: Metabolites with FC ≥ 2 (up-regulated) or ≤ 0.5 (down-regulated) are differential metabolites, as shown in Line 359-360.
In this study, the relative changes of metabolites of KR1326 (resistant cultivar) were observed with K1076(susceptible variety) as the control. The “up-regulated” represents that the content of this metabolite in resistant cultivar is higher than that in susceptible cultivar, while "down-regulated" means the opposite.
Point 22: It is very important to change graphs in figure 3 in a way to point out which metabolite changes are caused by resistance and susceptible C. melo root exudates – Fig 3B;C and D.
Response 22: At present, we can only screen out the differential metabolites, and can't analyze which type of metabolites are caused by differences in resistance and susceptibility, which requires further experimental analysis.
Point 23: All seven selected metabolites were down-regulated in Supplementary Table 3. I highly recommended prepare additional experiment to check how up-regulated metabolites will influence on haustorium induction rate. Moreover it will be worth to test how selected metabolites change weed germination.
Response 23: These suggestions are very meaningful to us. We will adopt your suggestions and conduct further experiments to supplement and improve these results of this study.
Discussion
I kindly suggest adding the following parts to the discussion:
Point 24: Is it known mechanism of resistance to P. aegyptiaca in C. melo cultivars? Could you discussed it in context of metabolomics data obtained in this study?
Response 24: As far as we know, there is no report on the resistance mechanism of C. melo to P. aegyptiaca. However, based on the information we have and the results in this study, we have discussed it in Line 575-582, The details are as follows: In addition, the allelochemicals secreted by the host are necessary for the germination and the formation of haustorium of parasitic plants. While some hosts with pre attachment resistance can make themselves "invisible" to parasitic plants by regulating metabolism and changing the type and content of allelochemicals secreted to the outside, or even directly secrete toxic chemcials to inhibit the germination and haustorium formation of parasitic plants, thus avoiding being parasitized[44]. Therefore, studying the metabolic differences of resistant and susceptible hosts is helpful to reveal the mechanism of host resistance and control parasitic plants.
Point 25: At which stage of C. melo development P. aegyptiaca is the most dangerous? Shouldn't exudates be collected in that time point to find novel haustorium-inducing factors (HIFs)?
Response 25: As far as we know, P. aegyptiaca can parasitize throughout the whole life cycle of the C. melo, including seedling, flowering, fruiting, and even after the host has died, P. aegyptiaca can still survive for a short time. In contrast, melon at flowering stage will absorb more nutrients to bear fruit. They will thrive and secrete more root exudates. Therefore, the root exudates of C. melo at flowering stage were collected. This question has been added into the second paragraph of the discussion.
Point 26: Two studied cultivars differ in plant size - especially in root morphology. Please discuss a little bit more how it could influence on metabolomic data obtain in this study.
Response 26: There must be differences between different cultivars, including KR1326 and K1076. plant size and root morphology are only phenotypic differences, which does not explain the difference in the haustorium induction effect of resistant and susceptible cultivars. This study is to explore the reason for the significant difference in the haustorium induction effect of the two cultivars from the plant metabolic aspects. And several HIFs have been screend eventually.
Point 27: Please discuss results from Miure et al., 2022 (Germination Stimulant Activity of Isothiocyanates on Phelipanche spp.) in context of metabolomics data obtained in your study as well as with your transcriptomic data Yao et al., 2016 (Global Transcriptomic Analysis Reveals the Mechanism of Phelipanche aegyptiaca Seed Germination).
Response 27: Thank you for your constructive suggestions. We have added the content of this article to the discussion.
Point 28: In future to study deeper role of HIFs during P. aegyptiaca parasitism, I will suggest validate role of found metabolites in A. thaliana - P. aegyptiaca model. It will be easy to find T-DNA mutants with impaired metabolomic pathways that have been described in your paper (for instance: Clarke et al., 2020 - Multiple immunity-related genes control susceptibility of Arabidopsis thaliana to the parasitic weed Phelipanche aegyptiaca).
Moreover described metabolites could be investigated with different parastitic plants from Orobanchaceae family.
Response 28: These suggestions are very meaningful to us. We will take your suggestions and further experiments will be conducted.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript “Screening of haustorium induction factors of Phelipanche aegyptiaca Pers. based on metabolome analysis of Cucumis melo L. root exudates” present the result of the experiment studying haustorium induction factors in the holoparasite Phelipanche aegyptiaca. The experiments are thoroughly done, and the results are very interesting and valuable. Although holoparasites have been studied for decades, HIFs of the obligate parasitic plant P. aegyptiaca are largely unknown. The authors have demonstrated that the root exudates of the host plant Cucumis melo L. contain allelochemicals displaying haustorium-inducing activity on P. aegyptiaca germinating seeds, and there are significant differences in the induction effects of the resistant and susceptible C. melo cultivars of P. aegyptiaca (KR1326 and K1076). The results provide clues for finding HIFs of obligate parasitic plants and shed new light on the control of P. aegyptiaca by regulating haustorium development.
However, few issues should be addressed to strengthen and clarify the manuscript.
1. latin names of some species should be italicized, for example, L16, 98, 122, 127, 130, and so on.
2. the family name should not be italicized, for example, L33, 59, 422, and so on.
3. L472 and 483 should be superscript.
4. L506: Write the full name of TFBQ and CPBQ.
Minor question:
1. L137-139: Have you standardized the root exudates with root biomass used for exudate collection? If not, how can you exclude mass effects?
2. Line377-379: what are the exact criteria for selection of chemicals for further examination?
Author Response
Please see below and in the attachment.
Response to Reviewer 2 Comments
Thank you for your attention and valuable comments on this manusprict. We attach great importance to your suggestions and make the following revision and replies:
Point 1: latin names of some species should be italicized, for example, L16, 98, 122, 127, 130, and so on.
Response 1: All Latin names have been checked, The Latin names in the L17, 66 , 99, 102, 128, 136, 452, 462 have been italicized.
Point2: the family name should not be italicized, for example, L33, 59, 422, and so on.
Response 2: The family name Orobanchaceae in the L35, 61, L35, 59, 452, 571, 596, 622 have been corrected.
Point3: L472 and 483 should be superscript.
Response 3: Ca2+ in L104, 10−4–10−6 in L518 have been corrected.
Point4: L506: Write the full name of TFBQ and CPBQ.
Response 4: The full name of TFBQ and CPBQ was Tetrafluorobenzoquinone and Cyclopropyl-p-benzoquinone respectively, which has been added to L546.
Point5: L137-139: Have you standardized the root exudates with root biomass used for exudate collection? If not, how can you exclude mass effects?
Response 5: We also took this problem into consideration when designing the experiment. First of all, the growth period and conditions of all melon plants are consistent, which minimizes the impact of external conditions on root exudates. In addition, to reduce the differences, we collected three groups of root exudates from 18 melon plants under the same culture conditions and mixed them into one replicate sample. Each cultivar has three replicates to reduce individual differences.
Point6: Line377-379: what are the exact criteria for selection of chemicals for further examination?
Response 6: At present, haustorium inducing factors are rarely reported in whole holoparasitic plants. We found the reported haustorium inducing factors which had significant influence in semi parasitic plants through reviewing the literature. The main reference sources are 12, 13, 14, 19, 20, 26, 28.
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript explores the germination pattern of holoparasite Phelipanche aegyptiaca under different metabolites and two host root exudates. However, the weakness of the study is reflected the incomplete experimental design, and these sections must be complete to give a reviewer confidence in the manuscript. Due to the failure to properly describe the experimental design, the masunscript should be improved under major revision. Specific comments below.
(1) lines 175-176: GR24 has been widely used in the seed germination of parasite, but why authors used it as a negative control.
(2) Unlike lateral haustorium, it is known that terminal haustorium should be formed sooner or later. Hence, I doubt if authors compared haustorium induction with different treatments only at the short period (7 day).
(3) lines 173-175: two cultivars represent resistant and susceptible types, so why authors mixed their root exudates as a positive control? I think two types should be used as positive and negative control, respectively.
(4) The resistant cultivar KR1326 and susceptible cultivar K1076 were selected to collect root exudates for seed germination and haustorium induction of P. aegyptiaca, it is interesting that the corresponding metabolites for these two cultivars may play key roles in improving or inhibiting haustoria formation.
(5) Figure 1 D, E, F: the standard error/deviation bar did not display significant differences between treatments, e.g. KR1326 and K1076 in fig. 1D, E, F.
Author Response
Please see below and in the attachment.
Response to Reviewer 3 Comments
Thank you for your attention and valuable comments on this manusprict. We attach great importance to your suggestions and make the following revision and replies:
Point 1: lines 175-176: GR24 has been widely used in the seed germination of parasite, but why authors used it as a negative control.
Response 1: Section 2.3 “Quantification of seed germination and haustorium induction” studies the effect of root exudates of KR1326 (resistant) and K1076 (susceptible) on seed germination and haustorium induction of P. aegyptiaca. In lines 175-176, we studied the effect of root exudates on haustorium formation rather than seed germination. GR24 has been widely used in the seed germination of parasite, but it cannot induce haustorium formation (our pervious experiments that GR24 induced seed germination have proved this conclusion). Therefore, GR24 was used as a negative control when studying the effect of root exudates on haustorium formation.
Point 2: Unlike lateral haustorium, it is known that terminal haustorium should be formed sooner or later. Hence, I doubt if authors compared haustorium induction with different treatments only at the short period (7 day).
Response 2: Firstly, as an obligate parasitie, P. aegyptiaca has a short independent survival period after germination in vitro. In addition, we have attempted to induce the haustorium of P. aegyptiaca for 20 days or more with adequate nutrition treatment , however, there was no significant difference between the number of haustorium formed at the later stage and that at 7th days. Therefore, the number of haustorium formed at 7th days was selected as the haustorium induction effect.
Point 3: lines 173-175: two cultivars represent resistant and susceptible types, so why authors mixed their root exudates as a positive control? I think two types should be used as positive and negative control, respectively.
Response 3: The positive control is required to evaluate the haustorium activity of different chemical compounds, while there is no reported haustorium induction factor of P. aegyptiaca at present. In this study, we can observe that the resistant and susceptible cultivars have significant haustorium induction activity from Figure 1F, so we choose root exudates as the positive control. However, metabolome analysis results in this study showed that there were differences between the metabolites in root exudates of the two cultivars. Mixed samples could collect as more HIFs as possible to comprehensively reflecting the induction effect of root exudates, and providing the reference for HIFs screening as control.
Point 4: The resistant cultivar KR1326 and susceptible cultivar K1076 were selected to collect root exudates for seed germination and haustorium induction of P. aegyptiaca, it is interesting that the corresponding metabolites for these two cultivars may play key roles in improving or inhibiting haustoria formation.
Response 4: After a long period of co-evolution of obligate parasite and their hosts, there are few host varieties with strong resistance. The resistant cultivar KR1326 and susceptible cultivar K1076 used in this study are the melon cultivar resistance identification results of pervious study (this article is under review and has not been published yet). The resistance of KR1326 is shown in the growth and development of P. aegyptiaca is blocked of early tubercle state and late unearthed stage compared with other melon cultivars, but it does not mean that resistant cultivar KR1326 have complete resistance to P. aegyptiaca. Therefore, we suggest that the root exudates of resistant and susceptible cultivar may contain metabolites that can improve or inhibit the haustorium formation of P. aegyptiaca. The purpose of this study is to find out the reasons for the differences of haustorium inducing activity between KR1326 and K1076 through analyzing the difference of metabolite content in root exudates, and to further find out the haustorium inducer factor of P. aegyptiaca in melon root exudates.
Point 5: Figure 1 D, E, F: the standard error/deviation bar did not display significant differences between treatments, e.g. KR1326 and K1076 in fig. 1D, E, F.
Response 5: The significant difference between KR1326 and K1076 in Figure 1 is the result of T-test using SPSS.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
No comments.
