Calcium signaling during the plant-plant interaction of parasitic Cuscuta reflexa with its hosts

The plant parasite Cuscuta reflexa induces various responses in compatible and incompatible host plants. The visual reactions of both types of host plants including obvious morphological changes require the recognition of Cuscuta ssp. A consequently initiated signaling cascade is triggered which leads to a tolerance of the infection or, in the case of some incompatible host plants, to resistance. Calcium (Ca²⁺) release is the major second messenger during signal transduction. Therefore, we have studied Ca²⁺ spiking in tomato and tobacco during infection with C. reflexa. In our recently published study¹ Ca²⁺ signals were monitored as bioluminescence in aequorin-expressing tomato plants after the onset of C. reflexa infestation. Signals at the attachment sites were observed from 30 to 48 h after infection. In an assay with leaf disks of aequorin-expressing tomato which were treated with different C. reflexa plant extracts it turned out that the substance that induced Ca²⁺ release in the host plant was closely linked to the parasite''s haustoria.Key words: cuscut, odder, calcium signaling, plant parasitismThe genera Cuscuta, also known as dodder, includes 170 parasitic species with a worldwide distribution. Members of Cuscuta ssp. belong to the 1% of angiospermic plants that live as holoparasites and depend on nutrients, water and carbohydrates from other host plants.² Cuscuta spp. lack roots or leaves but possess specific penetrating organs, the so called haustoria, which are fully developed 5–6 days after the first contact, when an interaction between parasite and host is established.As for all dicotyledonous plants, the typical Cuscuta spp. life cycle starts with the germination of seeds. At the stage of a rootless seedling, Cuscuta ssp. has just a few days to find and successfully invade a host plant. Although Cuscuta ssp. seedlings appear to coil indiscriminately around any vertical elongated object, they seem to have an efficient “sense of smell” to recognize potential “victims” and are therefore able to infest host plants more rapidly and efficiently.³ As soon as a host is reachable, Cuscuta ssp. starts to wind around the host shoot and initiates the attachment process as well as the development of haustoria.^2,4 Already at this initial phase of infection (12–48 h post attachment) the host plant senses the parasite and initiates an onset of several signals which are only partially known. Amongst the several induced genes are for example those encoding AGPs (Arabinogalactan Proteins), proteins promoting the parasite''s adherence.⁵ Also proteins are produced which might be important for nutrient and water uptake⁶ or which modify the host cell wall.⁷In this addendum article, we focus on signals which occur in host plants within the early infection stage prior to a vascular bundle connection and refer to our article about Ca²⁺ signalling in C. reflexa infected tomato plants.⁸ Besides phytohormones or other initial signalling molecules, such cellular calcium signals might be involved in controlling the expression of important genes for developmental or resistance related processes.In our approach, Cuscuta reflexa shoots of ∼25 cm length were wrapped around transgenic constitutively aeqourin-expressing tomato (Solanum lycopersicum) and tobacco (Nicotiana benthamiana) plants. With a highly sensitive ccd-camera we then monitored the two interacting organisms. The Ca²⁺-signals which are released by the host-plant could be detected as light-emission. The first cytosolic calcium signals were observed 24–48 h after the parasite attachment when the haustoria formation was already initiated. Light, indicating a cytosolic calcium influx was clearly visible directly where the parasite started to penetrate host tissue via its haustoria (Fig. 1) and often appeared several times within 1–6 h. As the light signals per recorded picture were collected for 10 min it is not clear if the duration of such cytosolic calcium influx comprises 10 ms or 10 min. An additional experiment in our study was the usage of a Cuscuta reflexa haustorium extract which was applied to aequorin expressing tomato leaf discs. Here it turned out that the Ca²⁺-ion influx happened steadily and slowly, because signals were only visible when summed up from 2 h recording. The finding that both boiled haustoria extract and control extract, made from Cuscuta reflexa shoots without haustoria, are inactive, suggests that a protein which is expressed during the infection process might be the direct or indirect trigger of such Ca²⁺-signals. These results overcome furthermore the theory that Calcium signals are induced by pressure, which might also be a step during Cuscuta ssp. infection.Open in a separate windowFigure 1Cuscuta reflexa infection induces calcium-signals in aequorin-expressing tomato. Left: Bright field; middle: light emission representing Ca²⁺-signals at the infection site ∼30 h post onset of the parasite; signals were monitored with a ccd camera. Right: overlay.The fact that calcium fluxes act as a second messenger in several stress responses such as cold shock, wind, touch, osmotic stress,⁹ phytohormone signalling pathways,¹⁰ plant—symbiotic interactions^10–12 or also plant pathogen interaction^13–15 complicates the interpretation of the signals that are induced by Cuscuta reflexa. One possibility could be that visible Ca²⁺-signals are part of a signalling pathway where also SA (salicylic acid) or/and JA (jasmonic acid) play an important role. Recently, Runyon et al.¹⁶ could show that tomato plants infected with Cuscuta pentagona respond with a strong induction of JA and SA 24–36 h post infections. This time frame correlates with our described calcium signals and it has been previously described that calcium fluxes might be a part of the JA- and SA-signalling cascade.The tomato—dodder interaction, however, represents an exception among dicotyledonous plants because tomato generates a hypersensitive response which is part of a successful resistance reaction.^7,16 In this particular case characteristic components of C. reflexa must be sensed by its host plant. These factors indicate “non-self” for the host plant, probably following a model comparable to the MAMP concept where characteristic molecular patterns of a pathogen are recognized in host plants via pattern recognition receptors and subsequently trigger defence responses.^17,18 But sensing and signalling in host plants takes place not only in the case of an “incompatible” interaction. The developmental phenomenons of a dodder—plant interaction in a “compatible” interaction are nearly a miracle. In this case, the parasite is completely tolerated and achieves the attachment and the penetration of the host plant. It interferes in developmental processes and manipulates its host to develop vascular tissues, to build up chimerical cell walls and interspecific symplastic cell connections.^16,17 Finally, it is connected to the host plant and starts to withdraw nutrients and carbohydrates^19,20 by mimicking endogenous sinks. Such a tolerated interaction reminds of an interaction of plants with bacterial or fungal symbionts, where also Ca²⁺-signals have been described and well characterized.^11,12 In the case of Cuscuta ssp.—host interaction a lot of further studies have to be done to discover all important steps of signalling cascades.