Molecular interaction among plants

4 May 2023 Dept. of Plant Biotechnology 1

Molecular Dialog Between Parasitic Plants and Their Hosts Upasana Mohapatra PALB-8092 II Ph. D. Plant Biotechnology UAS, GKVK, Bengaluru

CONTENTS Introduction Parasite identification of hosts Parasite–host exchange of RNAs Host Reaction to Attack by Root Parasitic Plants Casestudies Summary Future prospectiveses 4 May 2023 Dept. of Plant Biotechnology 3

INTRODUCTION Parasitism is a highly successful life strategy and a theme that bridges all kingdoms of life Parasitic plant: plant that receives all or part of its nutrients through a haustorial connection to another plant. • • Hemiparasite: Parasitic photosynthesize and thus is not completely dependent on the host plant for sugars Holoparasite: Parasitic plant that lacks the capacity for photosynthesis and thus relies entirely on uptake of sugars from host plants plant that can Facultative parasite: a parasitic plant that can complete its lifecycle as an autotrophic plant but retains the opportunistically plants. Facultative parasitic plants must be hemiparasitic 4 May 2023 Obligate parasites Must parasitize a host in order to complete their life cycles. Seeds of some obligate parasites require exposure to host signal molecules in order to germinate Clarke et al., 2019 Dept. of Plant Biotechnology capacity parasitize to host 4

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• In angiosperms, parasitism has independently evolved at least 12 or 13 times, and by recent estimates approximately 1% of parasitic. angiosperm species are Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 6

PARASITE IDENTIFICATION OF HOSTS Germination • Strigolactones (SLs) are the best-characterized class of germination stimulants • At least 20 different SL molecules have been identified in plants and plant families produce varying forms such that parasite seeds are able to differentiate among hosts on the basis of the identity of exuded SLs. Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 7

Parasitic plants use a protein related to D14 termed KARRIKIN INSENSITIVE 2 (KAI2; also known as HYPOSENSITIVE TO LIGHT) The Orobanchaceae KAI2 genes in parasitic expansion and specialization diverged KAI2 parasites (provides mechanism) to 1. Recognize specific host SLs, 2. Adapt to changes in host SL profiles, 3. Shift to recognize new hosts with a 4 May 2023 Dept. of Plant Biotechnology 8

Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 9

Locating the Host • Chemotropic growth of the parasite radicle in Orobanchaceae has been postulated for years 4 May 2023 Dept. of Plant Biotechnology 10

Yoshida & Shirasu, 2009 4 May 2023 Dept. of Plant Biotechnology 11

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Runyon et al., 2016 4 May 2023 Dept. of Plant Biotechnology 13

THE HAUSTORIUM AND ITS DEVELOPMENT • Haustoria are parasite-encoded multifunctional organs that attach the parasite to the host, penetrate into host tissues, and ultimately establish the physiological conduit through which resources flow between parasite and host Lateral haustoria • Develop on the sides of roots or stems, and terminal haustoria • More common cuscuta and most orobanchaceae • Develop on functioning roots without • Altering the tip meristem so multiple haustoria can develop on a single root Terminal haustoria • Develop on the tips of newly emerging radicles • More specialized • Obligate parasites • Alter the tip meristem so no additional root growth occurs until the haustorium has • Successfully invaded the host. Dept. of Plant Biotechnology 4 May 2023 14

Haustoria-inducing factors (HIFs): chemical cues released by host plants stimulating production of haustorial connections in parasitic plants First HIF identified Five general classes Flavonoids, Phenolic Acids, Quinones, Cytokinins Cyclohexene Oxides Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 15

• Phenolic acids become active HIFs only after oxidization to their sister quinones, haustorium induction may depend on the redox state of the inducer. • Subsequent intermediates formed during redox cycling between quinone and hydroquinone states of the HIF initiate a redox-sensitive signaling pathway leading to haustorium development experiments indicated that semiquinone Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 16

Not all Orobanchaceae respond to the same HIFs T. versicolor forms haustoria in response to DMBQ, but Triphysaria eriantha does not • Orobanche and Phelipanche do not form obvious haustorial structures in response to DMBQ They form haustoria when treated with root exudates of Brassica napus • Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 17

Attachment via Haustorial Hairs The first contact between certain Orobanchaceae parasites and hosts is made by haustorial hairs, which cement the parasite to the host When host and parasite roots were forced to grow closely together, haustorial hair mutants produced similar numbers of haustoria as do wild-type P. japonicum. Thus, haustorial hairs may play a role in host–parasite associations but not in haustorium initiation Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 18

Hormone action • As growth regulators of plant organogenesis, hormones play multiple roles in the development of haustoria. Major hormones are • Auxin • Ethylene • Cytokinin Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 20

Hormone action in haustoria development • Major hormones are • Auxin, • Ethylene • Cytokinin 2005 4 May 2023 Dept. of Plant Biotechnology 21

pHG8 YUC3-1 CONTROL 4 May 2023 Dept. of Plant Biotechnology 22

Viscum album on crabapple tree Malus toringoides 4 May 2023 Dept. of Plant Biotechnology 23

PARASITE–HOST EXCHANGE OF RNAs • Parasites exchange with their hosts’ proteins and RNAs that could contribute to shaping interactions between species. • A large number of C. campestris microRNAs are strongly expressed specifically at haustorial junctions. parasite growth and is therefore a potential strategy for crop improvement. • • Functional RNA-interference signals also move across parasitic plant haustoria. Hairpin transgenes in host plants trigger effective RNA interference in both T. versicolor and C. pentagona This host-induced gene silencing can be used to target parasite genes critical for • Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 25

(a) abiosis, the synthesis and release of cytotoxic compounds (e.g., phenolic acids, phytoalexins), by the challenged host root cells; Host Reaction to Attack by Root Parasitic Plants (b) rapid formation of physical barriers to prevent possible pathogen ingress and growth (e.g., lignification and other forms of cell wall modification at the host–parasite interface; Pre-attachment resistance includes all (a) no or reduced production of germination stimulant(s); (c) Release activation of programmed cell death in the form of a hypersensitive response at the point of parasite attachment development and retard its penetration; of reactive oxygen species and (b) production of germination inhibitors; to limit parasite (c) delay, reduction, or complete inhibition of haustorium formation leading to attachment incompetence; and (d) prevention of the parasite establishing the essential functional vascular continuity (i.e., xylem-to-xylem and/or phloem-to-phloem connections) with the host, delaying parasite growth followed by parasite developmental arrest and eventual death Dept. of Plant Biotechnology (d) development of preformed mechanical or structural barriers on the host surface to impede attachment. 4 May 2023 26 Gressel et al., 2013

An unsuccessful O. crenata penetration in root of a resistant vetch cultivar 20 days after inoculation, showing lignification of host cells, accumulation of a brown secreted material root showing a xylem vessel filled with mucilage 30 days after inoculation with O. crenata c,d- the haustorium is well developed with xylem continuity between parasite and host; in the resistant interaction, the haustorium invades the host root cortex but is not able to penetrate the endodermis to establish host– parasite xylem connectivity e- lignification of host tissue around the invading parasite f- the haustorium penetrates into the cortex but does not form connections with the host xylem Dept. of Plant Biotechnology 4 May 2023 27 Gressel et al., 2013

Parasitic plant and host interaction Clarke et al., 2019 4 May 2023 Dept. of Plant Biotechnology 28

Variable parasite growth form and mortality when grown with different hosts, suggest a dynamic and host-dependent molecular dialogue between the parasite and host 4 May 2023 Dept. of Plant Biotechnology 29

Host specificity and how it may restrict the distribution of hemi-parasitic plants in different plant communities along a steep ecological gradient. 4 May 2023 Dept. of Plant Biotechnology 30

Case study 1 1. Phytohormone performed to determine the responses of Cuscuta australis and its soybean (Glycine max) hosts to the feeding of green peach aphid (GPA; Myzus persicae) on C. australis. Investigated whether GPA feeding on Cuscuta induces local defense responses in the attacked Cuscuta plants, Whethrer Cuscuta sends systemic signals to the hosts, and if the systemic signals can elevate the resistance of hosts to subsequent insect attack. quantification, transcriptomic analysis and bioassays were 2. 3. 4 May 2023 Dept. of Plant Biotechnology 31

Materials and Methods Plants –grown in glass house insects – collected from a brassicaceous plant 10 cm GPA-infested C. australis effects on the transcriptomic and phytohormonal responses of its soybean host was studied Extraction and quantification of phytohormones RNA-seq and data analysis RT-qPCR GPA-infested C. australis effect on the insect resistance of its soybean host Detection of volatile-induced systemic responses in soybean plants Insect-infested soybean effect on GPA resistance of C. australis Dept. of Plant Biotechnology 4 May 2023 32

Cuscuta -Aphids Cuscuta + Aphids Soybean Soybean Treated Control Soybean aphids Soybean aphids 4 May 2023 Dept. of Plant Biotechnology 33

Conclusion • Decreased salicylic acid levels and 172 differentially expressed genes (DEGs) were found in GPA-attacked C. australis, and the soybean hosts exhibited increased jasmonic acid contents and 1015 DEGs, including > 100 transcription factor genes. • Importantly, GPA feeding on C. australis increased the resistance of the soybean host resulting in 21% decreased leafworm mass and 41% reduced aphid survival rate. • GPA herbivory-induced systemic signal from C. australis can elicit a strong transcriptomic reconfiguration in the soybean host. 4 May 2023 Dept. of Plant Biotechnology 36

Case study 2 Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets • Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. • In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants 4 May 2023 Dept. of Plant Biotechnology 38

Material and method CCD8sgRNA design and plasmid construction. Mutant verification and genotyping Analysis of off-target mutations. Evaluation of P. aegyptiaca resistance assay SL extraction and analysis using LC-MS/MS RNA isolation and quantitative real-time PCR. Carotenoid extract analysis. 4 May 2023 Dept. of Plant Biotechnology 39

Conclusion • Mutations in CCD8 can negatively affect tomato plant morphology; in a rootstock grafted to a wild-type scion, and might be combined with tomato rootstocks that are already resistant to fungal pathogens, viruses, and nematodes. • This method is expected to be effective against other Phelipanche and Orobanche species, if the parasite species share sufficient homology in their target sequences. • In addition, the mutated plants are devoid of foreign DNA sequences and are not considered genetically modified organisms 4 May 2023 Dept. of Plant Biotechnology 44

Summary • Development of the haustorium and its interactions with the host plant are becoming exciting areas of research, and new insights have emerged into its role in the exchange of hormones, nutrients, and macromolecules, including RNAs. • Transfer of mRNA and microRNA between host and parasite appears to be an important virulence and host adaptation strategy in Cuscuta. • Parallels are emerging between the molecular mechanisms mediating parasitic plant–host interactions and other plant– pathogen interactions, including the elicitation of host innate immunity. 4 May 2023 Dept. of Plant Biotechnology 45

Future prospectives 1. The processes by which germination factors and haustoria-inducing factors (HIFs) are secreted from host roots as well as their fate in the microbial-rich rhizosphere need to be better understood. 2. To clarify the importance of this novel virulence and adaptation strategy, researchers need to determine whether the transfer of microRNA and mRNA enhances parasite. 3. It is important to determine which damage- and parasite-associated molecular patterns as well as other elicitors of host plant immunity are present during host–parasite interactions and during which stages of parasitism the elicitors are present. 4. The presence and function of putative parasitic plant effector proteins should be studied across a wide range of parasitic plants to elucidate their host targets and the pathways they disrupt. 5. A critical issue—as yet unrealized—is to translate our gains in fundamental knowledge of parasitism toward deployment of effective parasitic weed control strategies 4 May 2023 Dept. of Plant Biotechnology 46

References • BARI, V.K., NASSAR, J.A., KHEREDIN, S.M., GAL-ON, A., RON, M., BRITT, A., STEELE, D., YODER, J. AND ALY, R., 2019. CRISPR/Cas9-mediated mutagenesis of CAROTENOID CLEAVAGE DIOXYGENASE 8 in tomato provides resistance against the parasitic weed Phelipanche aegyptiaca. Scientific reports, 9(1):1-12. CLARKE, C.R., TIMKO, M.P., YODER, J.I., AXTELL, M.J. AND WESTWOOD, J.H., 2019. Molecular Dialog Between Parasitic Plants and Their Hosts.Annual review of phytopathology, 57:279-299. GRESSEL, J., MUSSELMAN, L.J. AND JOEL, D.M. eds., 2013. Parasitic Orobanchaceae: Parasitic Mechanisms and Control Strategies. Springer Berlin Heidelberg. HETTENHAUSEN, C., LI, J., ZHUANG, H., SUN, H., XU, Y., QI, J., ZHANG, J., LEI, Y., QIN, Y., SUN, G. AND WANG, L., 2017. Stem parasitic plant Cuscuta australis (dodder) transfers herbivory-induced signals among plants. Proceedings of the National Academy of Sciences, 114(32):6703-6709. HONAAS, L.A., JONES, S., FARRELL, N., KAMEROW, W., ZHANG, H., VESCIO, K., ALTMAN, N.S., YODER, J.I. AND DEPAMPHILIS, C.W., 2019. Risk versus reward: host dependent parasite mortality rates and phenotypes in the facultative generalist Triphysaria versicolor. BMC plant biology, 19(1):334. RUNYON, J.B., MESCHER, M.C. AND DEMORAES, C.M., 2006. Volatile chemical cues guide host location and host selection by parasitic plants. Science, 313 (5795): 1964-1967. SPALLEK, T., MELNYK, C.W., WAKATAKE, T., ZHANG, J., SAKAMOTO, Y., KIBA, T., YOSHIDA, S., MATSUNAGA, S., SAKAKIBARA, H. AND SHIRASU, K., 2017. Interspecies hormonal control of host root morphology by parasitic plants. Proceedings of the NationalAcademy of Sciences, 114 (20):5283-5288. TOMILOV, A.A., TOMILOVA, N.B., ABDALLAH, I. AND YODER, J.I., 2005. Localized hormone fluxes and early haustorium development in the hemiparasitic plant Triphysaria versicolor. Plant Physiology, 138(3):1469-1480. ZHUANG, H., LI, J., SONG, J., HETTENHAUSEN, C., SCHUMAN, M.C., SUN, G., ZHANG, C., LI, J., SONG, D. AND WU, J., 2018. Aphid (Myzus persicae) feeding on the parasitic plant dodder (Cuscuta australis) activates defense responses in both the parasite and soybean host. New Phytologist, 218 (4):1586-1596. Zwanenburg, B., Pospíšil, T. and Zeljković, S.Ć., 2016. Strigolactones: new plant hormones in action. Planta, 243(6), pp.1311-1326. • • • • • • • • • 4 May 2023 Dept. of Plant Biotechnology 48

Molecular interaction among plants