Can plant developmental hormones affect microbiome assembly? And if so, how?

The plant hormone cytokinin (CK) is an important developmental regulator. Linking CK-mediated organ patterning to phyllopshere microbiome content, we recently found that CK shapes the phyllosphere microbiome, and that this underlies CK-mediated disease resistance.

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The abundance of different leaf forms in nature and, even in one particular habitat, can be astonishing. Why so many different leaf forms exist is a scientific question worthy of investigation, however, it always also seemed like a philosophical question to me.

Having a dual background in plant development and plant pathogen interactions, I was always fascinated by the idea that plant organ patterning could affect plant-microbe interactions. Since plant developmental hormones are the primary executors of organ development and patterning, we decided to examine their effect on microbial community content.

The plant hormone cytokinin (CK) is an important developmental regulator, promoting morphogenesis and delaying differentiation and senescence. From developmental processes, to growth, to stress tolerance, CKs are central in plant life. Previous work by us and others has demonstrated that CKs also mediate plant immunity and disease resistance (Fig. 1).

Fig 1: CK mediates disease resistance in tomato. The increased CK content transgene pBLS>>IPT7 and the increased CK sensitivity mutant clausa are more resistant to the gray mold fungal pathogen B. cinerea, while the reduced CK content transgene pFIL>>CKX is more sensitive. Image from Gupta et al., Mol Plant Pathol. 2020;00:1-20. doi: 10.1111/mpp.12978

In this work, we examined the effect of CK on the phyllosphere community, investigating both structural and chemical cues that could underlie its role in plant-microbe interactions. Here's what we found:

* CK drives phyllopshere microbial content

We used 16S rRNA sequencing to examine microbiome content in the CK-content mutants depicted in Fig. 1, as well as upon exogenous CK treatment. We found that CK poses a dominant effect on microbial community content in the phyllosphere, promotes microbial richness and diversity, and supports bacilli in the phyllosphere community (Fig. 2). We isolated bacilli from the phyllosphere of high CK genotypes, and found that they promote plant immunity and disease resistance (Fig. 3).


Fig 2: CK drives microbial content in the phyllosphere. The clustering of samples according to CK content in the genotype is much higher in High CK (pBLS>>IPT) or increased CK sensitivity (clausa) than in the background line (M82) or under low CK (pFIL>>CKX) (A). High CK genotypes (B) or exogenous CK treatment with three different CK derivatives (C) promote firmicutes, mostly bacilli, in the phyllosphere


Fig 3: Phyllosphere bacilli isolates from high CK genotypes promote disease resistance, exemplified here by lesion area in response to the gray mold pathogen B. cinerea, and immunity, exemplified here by the reactive oxygen species generated in response to the bacterial elicitor flg22. Gram positive bacilli isolates are indicated in red, and gram negative control bacteria, in blue

* CK-mediated immunity is partly dependent on the microbiome

Like in other eukaryotes, plant immunity was also reported to depend on the microbiome. Increased CK content or signaling genotypes, which are also disease resistant (Fig. 1), promote the formation of a protective epiphytic microbiome, by supporting bacilli that can inhibit disease progression (Fig. 2-3). To examine if CK-mediated immunity relies on these bacterial OTUs which it supports in the phyllosphere, we conducted disease experiments under sterile conditions, and found that CK-mediated disease resistance is significantly, though not completely, diminished, in sterile conditions (Fig. 4). The same was observed for the high CK genotypes, which are naturally more resistant to pathogen infection- a resistance which is diminished under sterile conditions.


Fig 4: CK mediated disease resistance depends on the microbiome. Gray mold disease levels are similar in Mock conditions in soil or cultured M82 plants, however, disease reduction mediated by CK treatment is significantly smaller under sterile conditions, leading to an increase in lesion areas upon CK treatment in sterile, cultured conditions, when compared with CK treatment under non cultured conditions.

* CK influences microbial content through structural and chemical cues

To examine how CK is affecting phyllosphere microbial content, we went back to the original question of organ patterning cues in microbiome formation, and examined the interaction of the leaf structure with the microbial community. To do this, we modeled leaves from the different genotypes in a biomimetic system, generating PDMS leaf imprints which were then used to generate agar leaf replicas that look like the natural leaves. The process used to generate the biomimetic leaves is detailed in the paper, and examples of these biomimetic leaf structures are provided in Fig. 5.


Fig 5. Leaf structural features captured in PDMS replicas. Light microscopy images of positive agar (A) and PDMS (B-D) replicas of leaves from the indicated genotypes. (A) Positive agar replicas alongside the natural leaf for reference. (B) Leaf adaxial topography including veins, bars= 500µM. (C) Recapitulation of leaf epidermal structure, including stomata, bars= 100 µM. (D) "Special" features are preserved in PDMS- placement of trichomes (M82), veins (pFIL>>CKX), trichomes (pBLS>>IPT), rugose topography (clausa). Bars= 500 µM, except in M82 where bar= 250 µM.  

We examined the dispersal of our bacterial isolates on the generated agar leaf replicas that contain the structural features of the genotypes from which they were modeled. We did so by spraying the replicas with bacterial solutions. Fig. 6 shows an example with one of the isolates. We found that several of our bacilli isolates showed a significant preference for the structures derived from high CK content or sensitivity genotypes, with the ratio between CFU growth on the structure to CFU growth off the structure being higher on structures mimicking high CK content or sensitivity genotypes (Fig. 6B). The gram negative isolates showed an opposite trend, preferring the structures derived from low CK genotypes, or exhibited no preference. The bacterial colonies also demonstrated altered morphology on the different structures, with the bacilli colonies being less round on the high-CK derived structures (Fig. 6 A,C).


Fig 6. Bacilli exhibit preference for synthetic leaf structures derived from High CK genotypes, and show decreased circularity on these structures. (A) Images depicting the colony morphology of B. megaterium 4C growing on the different genotype- derived synthetic leaf structures. (B) B. megaterium (OD600=0.01) was spray inoculated onto agar replicas of leaves of the indicated genotypes. Bacterial growth was quantified after 24 h. Preference or aversion to the leaf structure was assessed by quantifying the colony forming units (CFU) growing on the leaf structure, and dividing it by the CFU growing on an equal area in the surrounding structure-less agar. (C) Colony circularity was measured after 24 h using ImageJ.

We also examined a possible direct effect of CK on phyllosphere bacterial isolates, finding that the growth of some of them can be inhibited or enhanced by the presence of CK in vitro, providing another possible mechanism through which CK can affect microbial phyllosphere content.

 * CK may serve as a bidirectional signaling molecule in plant-microbe interactions

We found that the plant hormone CK acts as a selective force in microbial community formation, in part through its influence on organ structure, and that the microbiome partly underlies CK-mediated disease resistance. Our work dealt primarily with the host side in terms of CK effects on microbial colonization. Of course, it is well known that many different microbes can produce CKs. We did not investigate how microbial CK could be affecting plant-microbe interactions in the context of the phyllosphere. Given our results, we are now coming to think of CK as a core component in a holistic or circular system, where CK from both the plant host and the colonizing microbial strains could potentially act as a bidirectional signaling molecule, supporting a beneficial interaction between plant and microbe, and leading to increased vigor, preferential development, and disease resistance in the host, while simultaneously allowing the bacteria through their CK sensing system to identify preferred hosts for colonization.

Read the paper here https://doi.org/10.1038/s41396-021-01060-3

Maya Bar

Group Leader, ARO, Volcani Institute, Dept. of Plant Pathology