Host-parasite tissue adhesion by a secreted type of β-1,4-glucanase in the parasitic plant Phtheirospermum japonicum

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish intimate relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. Here we found that the model Orobanchaceae parasitic plant Phtheirospermum japonicum can be grafted on to interfamily species. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Despite different organs, we identified the shared gene expression profile, where cell proliferation- and cell wall modification-related genes are up-regulated. Among genes commonly induced in tissue adhesion between distant species, we showed a gene encoding a secreted type of β-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants.

The manuscript by Kurotani et al. has investigated the amazing potential of the parasitic plant Phtheirospermum japonicum to facilitate interspecific cell-to-cell adhesion between its haustorial cells and cells of its host. The authors showed that this ability is not limited to cells of the haustorium but that also the shoot is graft-compatible with a wider range of graft partners than typically observed for other species. They further describe the identification of a secreted glycosyl hydrolase (and 8 other proteins) with overlapping expression in graft and infection sites and provide evidence that this glycosyl hydrolase is involved in the establishment of the xylem bridge between parasite and host, an event that marks a successful parasitic relationship.
The well-written manuscript altogether provides a lot of convincing data that is well presented and that represents a significant step forward in our understanding of how parasitic plants can successfully attach to the vasculature of a broad host range. I have only found a few minor issues that should be addressed before this manuscript is ready for publication.
1. Page 8, lines 3, 9, and 26: Please check that Reference 20 (Honaas et al. 2013) in these lines is correct. It seems to me that it is Ref. 21 (Notaguchi et al. 2020) that was meant here. 2. Page 9: Regarding the knock-down PjGH9B3-mutant, I wonder whether the expression of the other GH9B3 family members is somehow affected? It would be relevant to know whether the knockdown is specific for one gene only or affected the other related genes as well, or whether other GH9B3 family members may take over the role of the knocked-down gene, possibly with some delay, see also comment 4 below. 3. Page 11, line 9: "haustorium" (correct the typo) 4. Page 11, discussion of the haustorium in the knockdown PjGH9B3: I wonder if the authors also analyzed older haustoria of the genetically modified (knockdown) line? Could it be that the knockdown just influences the efficiency of an infection and that after a longer period of time the same outcome (i.e. xylem bridge formation) is observed?
Reviewer #3 (Remarks to the Author): Brief summary of the manuscript The manuscript by Kurotani et al. describes the results of grafting Phtheirospermum japonicum, a model parasitic plant of the family Orobanchaceae, on species of a variety of plant families, among them and Arabidopsis. They show that P. japonicum can successfully be grafted, both as rootstock and scion on different species while it can parasitize achieving successful tissue adhesion and vasculature connection. Excessive grafting experiments show that viable grafts of P. japonicum scions on Arabidopsis stems can be produced. They further used 9 more species as rootstocks in grafting tests while P. japonicum was also used as a rootstock. Successful grafting was observed. Then the authors compare the transcriptome of grafted P.japonicum/Arabidopsis grafting regions with P. japonicum parasitism sites at Arabidopsis roots and interfamily grafting between Arabidopsis and Nicotiana, and they show that 9 genes are commonly induced in these three datasets. Detailed transcriptomic analyses of graft and parasitism interfaces revealed three clusters of commonly distinct expression patterns that shared GO terms. One of them, belongs to the family of Glycosyl hydrolase 9B family. GH9B3 from Nicotiana has been found before to be crucial for cell-cell adhesion in grafting. By expression analysis they show that this gene in P. japonicum follows the trend of other similar genes of the same plant family i.e. upregulation at the site of infection and after interfamily grafting. Knockdown of PjGH9B3 gene by RNAi experiments further suggest that this gene is possibly affecting the formation of xylem bridges between the host and the parasite, thus regulating infection process in a positive way.
Overall impression of the work My overall impression is that the present work is novel, complete, and all the experiments are well executed. The results are presented clearly. The study contributes significantly to the amount of knowledge we have on grafting and parasitism. It also presents important common aspects and mechanisms by which these two processes are functioning. I believe that it contributes significantly to basic scientific understanding although I would like to see some more practical applications (possibly on plant grafting).
Specific comments, with recommendations for addressing each comment 1. Lines 11 -14; this sentence needs re-writing. 2. Page 3; Lines 29 -Page 4; Lines 1; this sentence does not make sense and needs re-writing. 3. Page 8; Lines 5 -7. "Belonging" needs to be substituted with "Belongs". 4. Page 10; Line 1. "Realize" isn't a suitable verb here, maybe authors want to replace it with "materialize" or "implement" (heterografting). Fig. 4 where five homologous genes of P. japonicum are aligned to 2 Arabidopsis genes. Yet in the text (line 29) they mention four out of five homologous genes in the Orobanchaceae parasitic plants. Maybe they need to specify that it is specifically the P. japonicum genes in the alignment? Furthermore, in Figure 3b that depicts the expression patterns of genes after grafting and parasitism. Authors mention that genes PIN1, cyclin B1;2, PLL1, VND7 and OPS are all upregulated in a similar manner in both parasitism and grafting (genes that behaved similarly). Although I can see a similar pattern in the expression of OPS, VND7 and possibly PLL1, in PIN1 expression I see a reduction after 1 day of parasitism and then an increase (especially after the 2nd day) while in grafting there's an immediate increase in the expression after 1 day. Could the authors elaborate a bit more on why they interpret they results in this way?

Page 11; Lines 28 -30. Authors refer to the alignment of Supplementary
To Reviewer #1 (Remarks to the Author): In this MS, Kurotani et al. found that the hemiparasite, Phtheirospermum japonicum (PJ), could be grafted with inter-family species, as either the rootstock or scion. PJ was naturally (parasitization) or artificially grafted (stem-stem grafting) onto Arabidopsis, and samples from different times were collected and used for in-depth comparative transcriptome analysis. Even though many genes were similarly regulated in both sets of samples, the transcriptome profiles of PJ sample from parasitization and grafting experiment were largely distinct. The authors also show that a GH9B gene is commonly upregulated in PJ-Arabidopsis parasitization and grafting transcriptomes, as well as in the transcriptomes from Nicotiana-Arabidopsis grafting junction. Finally, the expression pattern of PjGH9B3 was determined using a PjGH9Be promoter-Venus reporter gene and RNAi of PjGH9B3 indicated that this glycosyl hydrolase gene, possibly a β-1,4-glucanase, is a positive regulator of parasitism.
This work nicely demonstrates the involvement of β-1,4-glucanases in parasitism, which have been shown to be involved in tissue adhension during artificial grafting, providing new insight into the mechanism underlying the establishment of parasitism.

I have only minor comments.
We thank the reviewer for considering our studies providing new insight into parasitism.
1. The RNA-seq samples were collected from the haustorial infestation and grafting regions. Thus, the RNA-seq data contain reads from both plant species, PJ and Arabidopsis. If I understood correctly, the reads were mapped to both genomes, and the Arabidopsis-specific reads and reads that could be mapped to both genomes were removed. The remaining reads were used for transcriptome analysis. If so, I would suggest adding a briefly description of how Arabidopsis transcripts were filtered out in the Results, so that the readers can better understand the transcriptome analysis.
ð Yes, the reviewer's understanding is correct. We described the filtering strategy of the sequence reads in Materials and Methods. We added a brief information also in Results (Page 6, Line 19-22).
2. One to 7 days and one to 14 days samples respectively from PJ parasitizing on Arabidopsis and PJ-Arabidopsis grafting were collected for RNA-seq. Why were different time windows used for these samples? The reasons should be indicated.
ð We appreciate your comment. We did not use the same timescale because observations of grafting between N. benthamiana and Arabidopsis and transcriptome analysis in previous study indicated that the time taken to establish grafting between distantly related plants was longer than the time taken to establish the parasitism.
We added a text to explain this in Results (Page 6, Line 15-16).
3. It is interesting that PJ can even function as a rootstock or scion and can be grafted with plants from other families. Being a rootstock or scion, PJ has different success rates during grafting and PJ cannot be grafted with Fabaceae species. The reasons could be discussed. 4. In the Abstract, "symbiotic" could be misleading, could be replaced with "parasitic".
ð Thank you. We corrected the text (Abstract, Line 3).
5. P8 L1-3. It is rather abrupt here that the transcriptomes from Nicotiana-Arabidopsis grafting system were used for further comparison. It would be better if the authors could elaborate the reasons.
6. P14 L9-11, it is important to know how the samples were collected. It would be better if the authors could indicate the details of sample collection, instead of citing other references. 7. P8 L9, the reference 20 is wrong.
To Reviewer #2 (Remarks to the Author): The manuscript by Kurotani et al. has investigated the amazing potential of the parasitic plant Phtheirospermum japonicum to facilitate interspecific cell-to-cell adhesion between its haustorial cells and cells of its host. The authors showed that this ability is not limited to cells of the haustorium but that also the shoot is graft-compatible with a wider range of graft partners than typically observed for other species. They further describe the identification of a secreted glycosyl hydrolase (and 8 other proteins) with overlapping expression in graft and infection sites and provide evidence that this glycosyl hydrolase is involved in the establishment of the xylem bridge between parasite and host, an event that marks a successful parasitic relationship.
The well-written manuscript altogether provides a lot of convincing data that is well presented and that represents a significant step forward in our understanding of how parasitic plants can successfully attach to the vasculature of a broad host range.
I have only found a few minor issues that should be addressed before this manuscript is ready for publication.
We thank the reviewer for considering our studies represents a significant step forward in understanding of parasitism to a broad host range. scions on Arabidopsis stems can be produced. They further used 9 more species as rootstocks in grafting tests while P. japonicum was also used as a rootstock. Successful grafting was observed.
Then the authors compare the transcriptome of grafted P.japonicum/Arabidopsis grafting regions with P. japonicum parasitism sites at Arabidopsis roots and interfamily grafting between Arabidopsis and Nicotiana, and they show that 9 genes are commonly induced in these three datasets. Detailed transcriptomic analyses of graft and parasitism interfaces revealed three clusters of commonly distinct expression patterns that shared GO terms.
One of them, belongs to the family of Glycosyl hydrolase 9B family. GH9B3 from Nicotiana has been found before to be crucial for cell-cell adhesion in grafting. By expression analysis they show that this gene in P. japonicum follows the trend of other similar genes of the same plant family i.e. upregulation at the site of infection and after interfamily grafting. Knockdown of PjGH9B3 gene by RNAi experiments further suggest that this gene is possibly affecting the formation of xylem bridges between the host and the parasite, thus regulating infection process in a positive way.
Overall impression of the work My overall impression is that the present work is novel, complete, and all the experiments are well executed. The results are presented clearly. The study contributes significantly to the amount of knowledge we have on grafting and parasitism. It also presents important common aspects and mechanisms by which these two processes are functioning. I believe that it contributes significantly to basic scientific understanding although I would like to see some more practical applications (possibly on plant grafting).
We thank the reviewer for considering our studies contribute significantly to the knowledge of grafting and parasitism. Since part of the mechanism of the establishment of grafting has been elucidated, together with the previously presented study, we believe that future improvements in practical grafting technology can be expected by utilizing these gene functions.