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Plants express numerous ‘pathogenesis-related’ (PR) proteins to defend themselves against pathogen infection. We recently discovered that PR-proteins such as chitinases, glucanases, peroxidases and thaumatin-like proteins are also functioning in the protection of extra-floral nectar (EFN) of Mexican Acacia myrmecophytes. These plants produce EFN, cellular food bodies and nesting space to house defending ant species of the genus Pseudomyrmex. More than 50 PR-proteins were discovered in this EFN and bioassays demonstrated that they actively can inhibit the growth of fungi and other phytopathogens. Although the plants can, thus, express PR-proteins and secrete them into the nectar, the leaves of these plants exhibit reduced activities of chitinases as compared to non-myrmecophytic plants and their antimicrobial protection depends on the mutualistic ants. When we deprived plants of their resident ants we observed higher microbial loads in the leaves and even in the tissue of the nectaries, as compared to plants that were inhabited by ants. The indirect defence that is achieved through an ant-plant mutualism can protect plants also from infections. Future studies will have to investigate the chemical nature of this mechanism in order to understand why plants depend on ants for their antimicrobial defence.
Plants fall victim to multiple attackers from various kingdoms and have evolved numerous strategies to defend themselves against herbivores and microbial pathogens. Pathogen resistance is usually based on particular cell wall properties, secondary compounds (phytoalexins) and on pathogenesis-related (PR) proteins.1,2 Plant resistance to pathogens is thus, mainly based on plant traits that interact with the pathogen and therewith functions as a “direct” defence mechanism. Resistance to herbivores, by contrast, can also be “indirect”: plants house or attract carnivores, which fulfil the defensive function.3 Defensive ant-plant interactions are common mutualisms in which plants provide to ants an array of rewards that comprises both food rewards and domatia (nesting space).3,4 Ants are attracted or nourished by plant-derived food rewards and defend the plants against herbivores.3,5 In obligate interactions, myrmecophytes are inhabited by specialised ants during major parts of their life4 and the ants are entirely dependent on the food rewards and nesting space that are provided by their host. These ants, in return, defend their host efficiently and aggressively against herbivores and encroaching vegetation. Only recently we found that this protective service might also cover the defence against phytopathogens.
In the Mexican Acacia-Pseudomyrmex mutualism, extrafloral nectar (EFN) represents an important plant trait to nourish symbiotic ant colonies. EFN is secreted by special glands called extrafloral nectaries and is rich in mono- and disaccharides and amino acids.6 Due to its high content in primary metabolites, EFN appears also attractive to exploiters, which make use of the nectar resource without providing a service to the plant.7 For example, EFN can serve as a suitable medium for pathogen growth and is highly prone to microbial infestations, which can have negative consequences on nectar composition.8,9
We recently found that Acacia EFN is biochemically protected from microbial infections.10,11 More than 50 proteins could be distinguished in EFN of the myrmecophytes, A. hindsii, A. cornigera and A. collinsii, and most of these proteins were annotated as PR-proteins such as chitinases, β-1,3 glucanases, thaumatin-like proteins and peroxidases. In fact, chitinases and glucanases represented more than 50% of the total amount of proteins in EFN of myrmecophytic Acacia plants.10,11 This dominance of PR-proteins clearly distinguishes the anti-microbial strategy of Acacia-EFN from the strategy of floral nectar of ornamental tobacco. The protection of the latter nectar is mainly based on small metabolites, such as hydrogen peroxide, which are produced by five proteins forming the ‘nectar redox cycle’.12–14 By contrast, PR-proteins have also been reported from pollination droplets of gymnosperms,15 although their biological functions remain to be studied. For the Acacia EFN, biotests demonstrated that the chitinases and glucanases are active and that EFN successfully can inhibit the growth of various fungi and oomycetes.11
Nevertheless, and although the microorganisms used in these assays were phytopathogens, we have now observed that the protection from microbial infection of the Acacia plant itself depends on certain characteristics of its ant inhabitants. The occurrence of fungi and bacteria in EFN and in the tissue of leaves and nectaries was investigated under natural growing conditions. We collected samples of A. cornigera and A. hindsii plants, which were inhabited by ants or which had been deprived of ants experimentally two weeks before. The tissues were extracted and analyzed for microbial infection by cultivating the extracts on malt agar plates, in order to quantify the abundances of life fungi and bacteria as the numbers of colony-forming units (CFU). Leaves of two species of myrmecophytic Acacia plants showed a significant increase in their bacterial load when they were deprived of the mutualistic P. ferrugineus ants (Fig. 1A). This observation is redolent of an earlier observation made on Macaranga myrmecophytes in Malaysia: lesions of these plants could easily be infected with fungi when the mutualistic Crematogaster ants were absent, but not in the presence of the ants.16 Similarly, myrmecophytic Piper plants depend, at least in part, on their ant inhabitants to obtain an efficient protection from fungal pathogens.17 Myrmecophytic Maracanga plants possessed reduced activities of chitinases in their leaves16 and the same phenomenon was found in Acacia myrmecophytes.18 Thus, the leaves of the obligate ant-plants of both genera, Acacia and Macaranga, appear not to express PR-proteins at sufficient activities as to protect themselves from pathogens and symbiotic ants are required for this defensive function. The effect is specific for the defending ants, because no significant differences between plants with and without ants could be detected in the case of plants that were inhabited by the non-defending parasite, P. gracilis19 (Fig. 1B). Most interestingly, even the nectary tissue appears to require ant-mediated protection from phytopathogens, whereas the EFN itself does not: no fungi can usually be isolated from freshly collected nectar of Acacia myrmecophytes under field conditions10 (Fig. 2A), but the tissue of the nectaries became heavily infected when branches were kept free of the defending ants (Fig. 2B).
Our new findings highlight the joint efforts of both ant and plant that are required for an efficient protection from pathogens of the myrmecophyte host plants, and, thus, for the prevention of this mutualism from exploitation by microorganisms. It remains open why the myrmecophytic Acacia plants exhibit reduced chitinase activities in their leaves18 and thus depend on ants for their antimicrobial protection, although they are fully equipped to express functioning PR-proteins and secrete them into their EFN.10,11 We could assume that an antimicrobial protection by ants is less costly for the plant than its own biochemical defence mechanisms. Alternatively, plant pathogens, which express multiple effectors in order to suppress their host resistance strategies,1 might be less capable to cope with ant-derived resistance mechanisms. Every attempt of an explanation, however, remains speculative as long as we do not understand how the ants can protect the leaves of their host plant from infection. The chemical mechanisms remain to be analyzed and may comprise both an ant-mediated resistance induction in the plant and directly ant-derived antimicrobial compounds. Most importantly, the joined efforts of both plant host and ant inhabitant are required to keep leaves, nectaries and nectar free of microbial infections and microbial pathogens have been identified as a further target of the indirect defence, which plants can obtain when they establish an obligate mutualism with ants.
Previously published online: www.landesbioscience.com/journals/psb/article/12038