Immune resistance in the red alga Dasysiphonia japonica against the oomycete pathogen Olpidiopsis heterosiphoniae

다국어 초록 (Multilingual Abstract)

Immune response is a response that organisms cause to defend against foreign invaders. Disease develops when the pathogen is able to evade the multiple layers of host defenses. Plants and animals, share striking similarities in their innate immune systems, some of which tell a story of likely convergent evolution. Red algae are earliest eukaryotes and also face diverse pathogens. Research on innate immunity of some red algae has been conducted, but it is limited to Pyropia yezoensis. Currently, more than 10,000 species of red algae have been reported, and more research is needed on the innate immunity of red algae. The Florideophyceae red algae Dasysiphonia japonica with more complex growth forms than Bangiophyceae red algae Pyropia yezoensis. Dasysiphonia japonica are differentiated filaments and the growth form consists of branched thallus.
The red alga Dasysiphonia japonica is infected by Olpidiopsis heterosiphoniae (collected from Korea), an oomycete, but the algae can coexist with the host for more than 5 years in culture, with the highest infection rate when first exposed to the pathogen (4 days) and infection decreasing over time, but still maintained. Infected D. japonica underwent some morphological and physiological changes. Fluorescent staining showed that infected filament showed reduced growth compared to control even when several cells were actually infected with the pathogen. The number of infected cells was decreased to about 40% after the six days of infection. Infected filament showed much higher tolerance to osmotic stress compared to control. The cell wall was more than twice thicker than control. Transcriptomic analysis was performed at 4- and 30-days after infection to investigate host's disease defense mechanisms that survive and are not dissipated from infection of the entire thallus by the pathogen. In total, 19,477 up-regulated and 905 down-regulated genes were identified from all infection groups. As a part of PAMP-triggered immunity (PTI), pathogen receptor genes (pattern recognition receptors), mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs) were found to be up-regulated after infection. As a part of the effector-triggered immunity (ETI), R protein domains (leucine rich repeat (LRR) and Toll/interleukin-1 receptor (TIR)) were found to be up-regulated after infection. Unlike P. yezoensis, genes related to hypersensitive apoptosis response (HR) was not observed, but cellulose synthase (CESA) was specifically up-regulated after infection. The results show that there is an innate immunity in D. japonica, and in case of O. heterosiphoniae infection, it increases the expression of the cellulose synthase gene to synthesize cellulose in the cell wall, thereby strengthening the cell wall to defend against infection by pathogens.
A new species Olpidiopsis pathogen infecting D. japonica was collected from Japan. The newly collected Olpidiopsis sp. are morphologically and molecularly distinct from O. heterosiphoniae. The infection pattern was also different, and the whole thallus were wasted within one month after infection. The two species were compared and analyzed to confirm the reason for the different infection patterns. Three cellulose synthase genes (DjCesA) were identified in D. japonica. Real-time PCR was conducted in both two genes. As a result, DjCesA3 was up-regulated after 2- and 4-days O. heterosiphoniae infection, but not in Olpidiopsis sp. infection. The results show that Olpidopsis sp. infection does not increase the expression of the cellulose synthase gene, which synthesizes cellulose in the cell wall, and thus does not strengthen the cell wall to defend against infection by pathogens.
After Olpidiopsis heterosiphoniae infection, reative oxygen species (ROS, stain by DCFH-DA) accumulated in the host cell membrane on 4-days infection, but was not observed in Olpidiopsis sp. infection. Four NADPH-oxidase genes (Djrboh) were identified in D. japonica. Real-time PCR was performed to confirm the expression of the rboh gene of D. japonica. As a result, Djrboh1-3 was up-regulated after 4-days O. heterosiphoniae infection, but not in Olpidiopsis sp. infection.
Our results reveal for the first time the existence of an innate immune response in Florideophyceae red algae Dasysiphonia japonica and confirm that NADPH-oxidase and cellulose synthase play important roles in defense against the Olpidiopsis pathogen. I expect that my results will serve as a starting point for understanding various disease defense mechanisms in red algae, which are still a mystery.

국문 초록 (Abstract)

면역 반응은 유기체가 외부 침입자에 대해 방어하기 위해 유발하는 반응이다. 질병은 병원체가 여러 층의 숙주 방어를 피할 수 있을 때 발생한다. 식물과 동물은 타고난 면역 체계에서 현저한 유사성을 공유하며, 일부는 수렴 진화를 이룬다. 홍조류는 최초의 진핵생물이며 또한 다양한 병원균에 직면한다.일부 홍조류의 선천면역 관련 연구가 진행되었으나 방사무늬김 (Pyropiayezoensis)에 한정된다. 현재 10,000종 이상의 홍조류가 보고되고 있으며, 홍조류의 선천면역에 대해서는 더 많은 연구가 필요하다. 홍조류는 크게 진정 홍조강 (Florideophyceae)과 원시홍조강 (Bangiophyceae)으로 나뉜다. 진정홍조강의 엇가지분홍풀은 (Dasysiphonia japonica)는 원시홍조강의 방사무늬김보다 더 복잡한 성장 형태를 가지고 있습니다. 엇가지분홍풀은 본질적으로 분화된 사상체이며 성장 시에는 분지된 엽체를 이룬다.
엇가지분홍풀은 난균류 (Oomycete)인 낭균 (Olpidiopsis heterosiphoniae,한국에서 채집됨)에 의해 감염되나, 5년 이상 숙주와 공존할 수 있으며, 최초병원체에 노출되었을 때(4일) 감염률이 가장 높다. 감염은 시간이 지남에 따라 감소하지만, 여전히 유지된다. 감염된 엇가지분홍풀은 형태학적 및 생리학적 변화를 겪었다. 세포벽염색 (Calcofluor White stain)을 통해 감염된 엽체의 감염되지 않은 세포를 포함하여 전체 엽체가 대조군에 비해 성장이 감소한것으로 나타났다. 감염된 세포의 수는 감염 6일 후에 약 40%로 감소하였다. 감염된 엽체는 대조군에 비해 삼투 스트레스에 대해 훨씬 더 높은 내성을 보였다. 세포벽은 대조군보다 2배 이상 두꺼웠다. 감염 후 4일 및 30일에 전사체 분석을 수행하여 병원체에 의한 전체 엽체의 질병 방어 메커니즘을 조사하였다. 총 19,477개의 상향 조절된 유전자와 905개의 하향 조절된 유전자가
모든 감염 그룹에서 확인되었습니다. PAMP 유발 면역(PTI)의 일부로서 병원체 수용체 유전자(패턴 인식 수용체), MAPK(mitogen-activated protein kinase) 및 CDPK (calcium-dependent protein kinase)가 감염 후 상향 조절되는 것으로 밝혀졌다. ETI (Effector-Triggered Immunity)의 일부로서 R 단백질 도메인 (LRR (leucine rich repeat) 및 TIR (Toll/interleukin-1 receptor))이 감염 후 상향 조절되는 것으로 밝혀졌다. 방사무늬김과 달리 과민성 세포사멸 반응(HR)과 관련된 유전자는 관찰되지 않았지만, 셀룰로오스 신타아제(CESA)는 감염 후 특이적으로 상향 조절되었다. 이상의 결과는 엇가지분홍풀에 선천면역이 존재하고, 낭균 (O. heterosiphoniae) 감염의 경우 세포벽에서 셀룰로오스를 합성하는 셀룰로오스 합성효소 유전자의 발현을 증가시켜 세포벽을 강화시켜 병원체의 추가적인 감염을 저해시키는 것으로 사료된다.엇가지분홍풀을 감염시키는 새로운 낭균 (Olpidiopsis sp.) 병원체가 일본에서 채집되었다. 새로 채집된 Olpidiopsis sp. 는 형태적으로 O.heterosiphoniae 와 형태학적으로나 분자적으로 구별된다. 또한, 감염 후 1개월 이내에 엽체 전체가 감염에 의해 소진되었다. 감염 패턴이 다른 이유를 확인하기 위해 두 종을 비교 분석했다. 낭균 감염 후 세포벽의 셀룰로스의 변화를 알아보기 위해 총 3개의 엇가지분홍풀의 Cellulose synthase 유전자(DjCesA)를 확인하였다. Real-time PCR를 통해 두 종의 낭균 감염 후 감염 시간별로 DjCesA1-3 의 발현을 확인하였다. 그 결과, DjCesA3 는 O.heterosiphoniae 감염 2일 및 4일 후에 상향 조절되었지만 Olpidiopsis sp.감염 후 DjCesA1-3 는 모두 뚜렷한 변화를 보이지 않았다.질병 방어에서 중요한 신호전달 물질인 활성산소가 낭균 감염 후 생성되는지 확인하기 위해 활성산소종 염색 (DCFH-DA)을 수행하였다. O.heterosiphoniae 감염 4일째 활성산소가 숙주 세포막에 축적되었으나Olpidiopsis sp.에서는 활성산소 염색이 관찰되지 않았다. 엇가지분홍풀의 전사체 분석에서 총 4개의 세포막의 활성산소를 생성하는 효소인NADPH-oxidase 유전자를 확인하였다. 엇가지풀의 NADPH-oxidase 유전자 (Djrboh1-4)가 두 종의 낭균 감염 후 감염 시간별로 어떻게 발현하는지확인하기 위해 Real-time PCR을 수행하였다. 그 결과, Djrboh1-3 은 O.heterosiphoniae 감염 4일 후에 상향조절되었지만 Olpidiopsis sp. 의 감염에서는 Djrboh1-4 모두 뚜렷한 변화를 보이지 않았다.이상의 결과는 진정홍조강의 엇가지분홍풀에서 선천적 면역 반응의 존재를처음으로 밝혔고 NADPH-oxidase와 cellulose synthase가 낭균성 병원체의감염에서 중요한 역할을 한다는 것을 확인하였다. 아직 연구가 미비한 홍조류의 다양한 질병 방어 기전을 이해하는 출발점이 되기를 기대한다.

목차 (Table of Contents)

• Chapter Ⅰ. General introduction 1
• 1.1. Species of algae oomycete pathogens and their host specificity 2
• 1.2. Innate immunity of algae against pathogen 3
• 1.3. Aim of this study 4
• CHAPTER Ⅱ. Oomycete disease of red algae Dasysiphonia japonica causing by pathogen Olpidiopsis heterosiphoniae 12
• 2.1. Morphological and physiological changes in Dasysiphonia japonica after Olpidiopsis heterosiphoniae infection 13
• 2.2. Transcriptome researches of Dasysiphonia japonica after Olpidiopsis heterosiphoniae infection 29
• CHAPTER Ⅲ. Comparative analysis of the regulation of NADPH-oxidase and cellulose synthase in Dasysiphonia japonica after Olpidiopsis spp. infection 54
• 3.1. New oomycete pathogen Olpidiopsis sp. that infect Dasysiphonia japonica 55
• 3.2. Comparative analysis of cellulose synthase (CESA) in Dasysiphonia japonica after Olpidiopsis spp. infection 66
• 3.3. Comparative analysis of NADPH-oxidase (RBOH) in Dasysiphonia japonica after Olpidiopsis spp. infection 77
• Concluding remarks 95
• References 98
• Abstract in Korean 108

참고문헌 (Reference)

1 Sparrow, F. K., "Aquatic Phycomycetes, ed. 2", 1187 pp, 1960

2 Canter, H. M., "On Myzocytium megastomum De Wildeman", 31: 80–IN8, 1947

3 Raghukumar, C., "Fungal parasites of the green alga Chaetomorpha media", 50: I00, 1987

4 Sparrow, F. K., "Observations on marine Phycomycetes collected in Denmark", 8: 1–24, 1934

5 Dixon, P. S., "Studies on marine algae of the British Isles: the genus Ceramium", 39: 331–374, 1960

6 Archibald, J. M., von Dahlen, J. K., de Vries, S., de Vries, J., Gould, S. B., "On plant defense signaling networks and early land plant evolution", 11: 1–14, 2018

7 Bresnan, E., Perrineau, M. M., Garvetto, A., Gachon, C. M., Dressler‐Allame, M., "Ectrogella parasitoids of the diatom Licmophora sp. are polyphyletic", 67: 18– 27, 2020

8 Jermiin, L. S., Wong, T. K., Von Haeseler, A., Minh, B. Q., Kalyaanamoorthy, S., "ModelFinder: fast model selection for accurate phylogenetic estimates", 14: 587–589, 2017

9 Bowman, J. L., Tan, Q. W., Mutwil, M., Lampugnani, E. R., Flores-Sandoval, E., "Cellulose synthesis–central components and their evolutionary relationships", 24: 402-412, 2019

10 Petersen, H. E., "Contributions a la connaissance des phycomycetes marins (Chytridinae Fischer)", 5: 439–488, 1905

11 Chandramohan, D., Raghukumar, C., "Changes in the marine green alga Chaetomorpha media on infection by a fungal pathogen", 31: 311–315, 1988

12 Kim, G. H., Kwak, M. S., Klochkova, T. A., "A new endoparasite Olpidiopsis heterosiphoniae sp. nov. that infects red algae in Korea", 28: 264–269, 2017

13 Johnson, L. M., Whisler, H. C., Tuininga, A. R., Kerwin, J. L., "Infection and morphogenesis of Pythium marinum in species of Porphyra and other red algae", 70: 1017–1024, 1992

14 Jung, S., Klochkova, T. A., Kim, G. H., "Host range and salinity tolerance of Pythium porphyrae may indicate its terrestrial origin", 29: 371–379, 2017

15 Badis, Y., Strittmatter, M., Murua, P., Klochkova, T. A., Garvetto, A., "Novel species of the oomycete Olpidiopsis potentially threaten European red algal cultivation", 31: 1239 –1250, 2019

16 Takahashi, M., "Pythium porphyrae Takahashi et Sasaki, sp. nov. causing red rot of marine red algae Porphyra spp", 18: 279–285, 1977

17 Aleem, A. A., "Olpidiopsis feldmannii sp. nov., Champignon marin parasite d'Algues de la famille des Bonnemaisoniacees", 235: 1250–1252, 1952

18 Baumeister, T. U., Vallet, M., Kaftan, F., Grabe, V., Buaya, A., "The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom", 10: 4938, 2019

19 Ausland, C., Orton, L., Grayburn, W. S., Fitzek, E., Entwistle, S., "Cell wall enzymes in Zygnema circumcarinatum UTEX 1559 respond to osmotic stress in a plant-like fashion", 10: 732, 2019

20 Buaya, A. T., Thines, M., "Diatomophthoraceae–a new family of Olpidiopsis-like diatom parasitoids largely unrelated to Ectrogella", 5: 113–118, 2020

21 Brurberg, M. B., Roleda, M. Y., Ojeda, D. I., Herrero, M. L., "Occurrence and pathogenicity of Pythium (Oomycota) on Ulva species (Chlorophyta) at different salinities", 35: 79–89, 2020

22 Badis, Y., Klochkova T. A., Kim G. H., Han, J. W., Gachon C. M. M., "The gene repertoire of Pythium porphyrae (Oomycota) suggests an adapted plant pathogen tackling red algae", 35: 133–144, 2020

23 Crowell, E. F., Pochylova, Z., Juraniec, M., Jouy, H., Desprez, T., "Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana", 104: 15572–15577, 2007

24 Anisimova M, Lefort V, Hordijk W, Guindon S, Dufayard JF, "New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0", 59: 307–321, 2010

25 Aleem, A. A., "Phycomycetes marins parasites de diatomees et d'algues dans la region de Banyuls-sur-Mer (Pyrenees-Orientales)", 4: 421–440, 1950

26 Antolić, B., Tsirigoti, A., Nikolić, V., Katsaros, C., Fletcher, K., "New record and phylogenetic affinities of the oomycete Olpidiopsis feldmanni infecting Asparagopsis sp. (Rhodophyta)", 117: 45–57, 2015

27 Beakes, G. W., West, J. A., Sekimoto, S., Klochkova, T. A., Honda, D., "Olpidiopsis bostrychiae sp. nov.: an endoparasitic oomycete that infects Bostrychia and other red algae (Rhodophyta)", 48: 460–472, 2009

28 Blouin, N. A., Wheeler, G. L., Lohr, M., Ficko-Blean, E., Brawley, S. H., "Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta)", 114: E6361– E6370., 2017

29 Pereira, S., Velez, C., "Live observations on Myzocytium megastomum (Lagenidiales), parasitizing a green alga, Rhizoclonium sp. (Siphonocladales)", 78: 469–474, 2004

30 Ball, S. G., Porcel, B., Collen, J., Chaparro, C., Carre, W., "Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida", 110: 5247–5252, 2013

31 Buaya, A. T., Thines, M., "Miracula moenusica, a new member of the holocarpic parasitoid genus from the invasive freshwater diatom Pleurosira laevis", 3: 35–40, 2019

32 Han, J. W., Zuccarello, G. C., Shim, J., Shim, E., Kim, G. H., "Keeping house: evaluation of housekeeping genes for real-time PCR in the red alga, Bostrychia moritziana (Florideophyceae)", 31: 167–174, 2016

33 Buaya, A. T., Thines, M., Scholz, B., "Sirolpidium bryopsidis, a parasite of green algae, is probably conspecific with Pontisma lagenidioides, a parasite of red algae", 7: 223–231, 2021

34 Dick, M. W., "The species and systematic position of Crypticola in the Peronosporomycetes, and new names for Halocrusticida and species therein", 102: 1062–1066, 1998

35 Buaya, A. T., Thines, M., Scholz, B., "Taxonomy and phylogeny of Aphanomycopsis bacillariacearum, a holocarpic oomycete parasitoid of the freshwater diatom genus Pinnularia", 20: 289–298, 2021

36 Gachon, C. M., Strittmatter, M., Müller, D. G., Küpper, F. C., Kleinteich, J., "Detection of differential host susceptibility to the marine oomycete pathogen Eurychasma dicksonii by real-time PCR: not all algae are equal", 75: 322– 328, 2009

37 Buaya, A. T., Thines, M., Ploch, S., "Rediscovery and phylogenetic placement of Olpidiopsis gillii (de Wildeman) Friedmann, a holocarpic oomycete parasitoid of freshwater diatoms", 60: 141–146, 2019

38 Hwang, M. S., Shim, J. B., Klochkova, T. A., Kim, G. H., "Host –parasite interactions and host species susceptibility of the marine oomycete parasite, Olpidiopsis sp., from Korea that infects red algae", 24: 135–144, 2012

39 Badis, Y., West, P. V., Strittmatter, M., Gachon, C. M., Fletcher, K. I., "Pathogens of brown algae: culture studies of Anisolpidium ectocarpii and A. rosenvingei reveal that the Anisolpidiales are uniflagellated oomycetes", 52: 133–148, 2017

40 Ehrhardt, D. W., Paredez, A. R., Lindeboom, J. J., Gutierrez, R., Emons, A. M. C., "Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments", 11: 797–806, 2009

41 Karling, J. S., "The life history of Anisolpidium ectocarpii gen. nov. et sp. nov., and a synopsis and classification of other fungi with anteriorly uniflagellate zoospores", 637–648, 1943

42 Anwar, M., Zhao, D., Mei, R., Lan, C., Guo, C., "The Functionally Characterization of Putative Genes Involved in the Formation of Mannose in the Aplanospore Cell Wall of Haematococcus pluvialis (Volvocales, Chlorophyta)", 11: 725, 2021

43 Kupper, F. C., Muller, D. G., Kupper, H., "Infection experiments reveal broad host ranges of Eurychasma dicksonii (Oomycota) and Chytridium polysiphoniae (Chytridiomycota), two eukaryotic parasites in marine brown algae (Phaeophyceae)", 47: 217–223, 1999