Microbial CKs mediate G20-18 biocontrol

Since the CK-producing PGP Pfl strain G20-18 had not been tested for its biocontrol abilities, we first examined its biocontrol potential in the Arabidopsis–Pto pathosystem26 in comparison to its CK-deficient transposon mutants CNT1 and CNT224,25. As CKs have been demonstrated to induce defence responses or resistance against (hemi)biotrophic foliar pathogens when applied to leaves of Arabidopsis10,11,27, rice13,16 and tobacco12,28, we decided to analyse the biocontrol potential of the Pfl strains when directly applied to Arabidopsis leaves by infiltration of cell suspensions 48 h prior to Pto infection. The leaf infiltration assay widely used in model pathosystems was chosen to allow us to relate the findings to the well-established immunity-relevant CK functions in leaf tissues. Although approaches such as spray inoculation or application to the root system would address more natural scenarios of interaction, they would contribute additional sources of interference with CK-mediated immunity responses and thus, further complicate the analyses of a potential role of CK in biocontrol.

Pre-treatment with Pfl G20-18 heavily suppressed Pto symptom development at 4 days post infection (dpi), resulting in maintenance of tissue integrity, an important beneficial aspect of biocontrol applications in sustaining biomass yield. Mock pre-treatment had no effect on Pto symptoms compared to control infections without pre-treatment (Fig. 1a). Thus, G20-18 is considered an efficient strain for biocontrol of Pto in Arabidopsis in the leaf infiltration assays. In comparison to G20-18, both CNT transposon mutants had only a slight suppressive effect on Pto symptom development (Fig. 1a). The quantification of the average symptom scores over all experiments further demonstrates this biocontrol effect: G20-18 pre-treatment efficiently suppressed Pto symptoms by approximately 75%, CNT pre-treatments suppressed Pto symptoms only by 15 to 20% compared to untreated and mock controls, indicating that the CK-deficient mutants were significantly less effective than G20-18 (Fig. 1b). This highly reduced effect of the CK-deficient CNT transposon mutants on Pto symptom development strongly supports a role for microbial CK production in the biocontrol ability of G20-18.

Figure 1 Pfl G20-18 suppresses Pto symptoms in Arabidopsis. (a) Pto symptom development in Arabidopsis leaves (right halves) 4 days post infection (dpi) with 106 cfu ml−1 is strongly suppressed by G20-18 compared to controls and CNT pre-treatments. (b) Average Pto symptom score in Arabidopsis 4 dpi with 106 cfu ml−1 is significantly lower after G20-18 pre-treatment compared to controls and CNT pre-treatments. Data are means ± s.e. n ≥ 300, letters indicate different significance groups (P < 0.05). Full size image

As the CNT transposon mutants were generated by undirected mutagenesis via the introduction of the TnphoA transposon into G20-18 and were selected based on CK deficiency without detailed genetic characterization24, we analysed the only known CK biosynthetic gene in Pfl strains, tRNA delta(2)-isopentenylpyrophosphate transferase (miaA). Using primers based on known Pfl miaA sequences the gene was amplified from G20-18 and sequenced (Supplementary Fig. 1). Size comparison of full-length miaA amplicons of G20-18 and the CNT transposon mutants as well as sequence analysis ruled out miaA as the direct target of TnphoA. Subsequent semi-quantitative RT-PCR analysis revealed that miaA transcript levels in the CNT transposon mutants were strongly reduced by approximately 50% compared to G20-18 (Supplementary Fig. 2). This suggests that regulatory components in the CNT mutants were affected by the transposon mutagenesis, potentially interfering with miaA transcription or the processing and stability of miaA transcripts. Since the mechanism of transcriptional regulation of miaA is not elucidated, we used directed functional approaches to further substantiate the link between miaA as a determinant of microbial CK production and subsequent biocontrol activity against Pto.

Considering the reduced miaA transcript levels in the CNT transposon mutants, compared to G20-18, as the cause for the difference in biocontrol efficacy, functional complementation for CK production by the CNT transposon mutants (gain-of-function) was performed to assess the possible restoration of their biocontrol ability. Therefore, the CK biosynthetic genes isopentenyltransferase from Agrobacterium tumefaciens (ipt) for heterologous expression and the endogenous Pfl G20-18 miaA for homologous expression were fused to a lac-promoter in the expression vector pBBR1MCS-5. The different Pfl strains were transformed with these gain-of-function constructs or the empty vector (EV) and analysed for their biocontrol activities. The presence of the EV did not affect biocontrol activity of G20-18 as this strain efficiently restricted Pto symptom development (Fig. 2a) comparable to G20-18 wild-type (Fig. 2b). Also in the CNT transposon mutants, the EV did not cause changes (Fig. 2a) as symptoms were still significantly less suppressed compared to G20-18 (Fig. 2b). In contrast, the ipt- or miaA-complemented CNT transposon mutants, exhibited restored biocontrol activities as evidenced by a strong suppression of Pto symptom development (Fig. 2a), comparable to G20-18 biocontrol activity (Fig. 2b). This wild-type-like biocontrol activity in the two CNT transposon mutants functionally complemented via restored CK production by two different CK biosynthetic genes supports the role of microbial CKs as a key determinant for efficient biocontrol of Pto.

Figure 2 Complementation of the CNT transposon mutants with a functional CK biosynthetic gene restores their biocontrol ability. (a) The biocontrol ability of CNT transposon mutants is restored by complementation with functional Atipt or G20-18miaA evident from strongly reduced Pto symptoms (right leaf halves) 4 days post infection (dpi) with 106 cfu ml−1. Transformation with the empty vector pBBRMCS-5 (EV) has no effect. (b) Average Pto symptom score in Arabidopsis 4 dpi with 106 cfu ml−1 after indicated pre-treatments. Data are means ± s.e. n ≥ 226, letters indicate different significance groups (P < 0.05). Full size image

To substantiate the gain-of-function data, a complementary loss-of-function approach was followed, addressing the function of miaA and subsequent CK production in G20-18-mediated biocontrol of Pto. To this end, the impact of directed knockout of the G20-18 miaA gene by insertion of a kanamycin resistance cassette into the miaA coding region on the biocontrol ability was assessed. This resulted in the Pfl knockout mutant ΔmiaA, which tested PCR-positive for the integration of the disrupted miaA gene sequence in its genome. RT-PCR confirmed the lack of miaA transcripts and thus the functional knockout in this strain (Supplementary Fig. 2). Assays with this ΔmiaA knockout mutant revealed a significant reduction in biocontrol compared to G20-18 wild-type as illustrated by stronger Pto symptom development (Fig. 3). Together, the gain-of-function and directed loss-of-function approaches prove the importance of microbial CK production for their biocontrol ability in the leaf infiltration assays. Interestingly, the distinct functional miaA knockout in ΔmiaA (Supplementary Fig. 2) did not further reduce the biocontrol ability compared to the transposon mutants CNT1 and 2 (Fig. 3) in which low levels of miaA transcripts were still detectable (Supplementary Fig. 2). This suggests that the described biocontrol effect depends on minimum threshold levels of miaA transcripts which subsequently determine CK levels that suffice to induce resistance under particular conditions.

Figure 3 Distinct ∆miaA knockout in Pfl G20-18 exhibits a reduced biocontrol activity. (a) ΔmiaA loss-of-function mutant is impaired in its biocontrol ability indicated by stronger Pto symptom development (right leaf halves) 4 days post infection (dpi) with 106 cfu ml−1 compared to Pfl G20-18 pre-treatment. (b) Average Pto symptom score in Arabidopsis 4 dpi with 106 cfu ml−1 after indicated pre-treatments. Data are means ± s.e. n ≥ 79, letters indicate different significance groups (P < 0.05). Full size image

G20-18 biocontrol affects CKs in planta

Based on the established link between Pfl G20-18 CK production and its biocontrol abilities described above, the in planta CK levels were analysed as these should ultimately reflect their contribution to the induction of resistance or defence responses10,11,12,13,14,15,27. Therefore, we analysed the accumulation of 25 individual CK species comprising the free nucleobases as well as conjugates29 in pooled samples of whole Arabidopsis leaves 48 h post infiltration with the different Pfl strains, which corresponds to the time-point of Pto infection. Thus, these samples integrate all processes related to each individual pre-treatment and determine the plant tissue status at the critical time-point of infection that defines the outcome of the plant-pathogen interaction. CKs were analysed in two sample sets, one comparing the pre-treatments with G20-18, the miaA- or ipt-complemented CNT transposon mutants and mock control (Table 1 and Supplementary Table 1) and the second comparing pre-treatments with G20-18, the CNT transposon mutants, the ΔmiaA knockout mutant and mock control (Table 1 and Supplementary Table 2). Eight of ten CK levels that increased after G20-18 treatment in the first set (Supplementary Table 1) also increased in the second set (Supplementary Table 2). A clear trend of lower CK levels in plant tissue pre-treated with loss-of-function CNT transposon or ΔmiaA knockout mutants was observed compared to G20-18 (ratios of 0.82 to 0.89). In contrast, this effect was reversed in tissue treated with the functionally complemented CNT transposon mutants that showed even higher CK levels compared to G20-18 (ratios of 1.08 to 1.11, Table 1). Since CK types differ in their biological activity and signalling function, the individual consideration of specific CK species is important. Total tZ-, cZ-, DHZ- and iP-type CK levels showed similar trends as total CK levels with lower levels after treatments with the CK-deficient mutants (Supplementary Table 2) and reversion in the functionally complemented CNT transposon mutants (Supplementary Table 1), which correlates with their differential effect on Pto symptom development (Fig. 1, 2, 3). Similarly, levels of the free nucleobases as the most active CK species5 were lower in tissue treated with CK-deficient mutants (ratios of 0.63 to 0.97) and higher in tissue treated with the functionally complemented CNT transposon mutants (ratios of 1.34 to 2.38) compared to G20-18 (Table 1). In particular, the individual nucleobases tZ, cZ and iP accumulated to higher levels after treatment with the functionally complemented CNT transposon mutants compared to G20-18 (ratios of 1.30 to 2.50), while these nucleobases in general showed lower accumulation after mutant treatments (Table 1). The most prominent differences were detected in the accumulation of the highly active tZ. Treatment with the three CK-deficient mutants caused significantly lower tZ levels (ratios of 0.47 to 0.60) than G20-18 treatment (Table 1), which can directly be related to the defects in miaA expression as miaA has been identified to be responsible for the specific formation of tZ and derivatives from tRNA in different bacteria30,31,32. Intriguingly, exogenously supplied tZ efficiently restricted infections of Arabidopsis with Pto10 and tobacco with P. syringae pv. tabaci (Pst)12,28, while cZ had a much weaker effect on the resistance against Pst28 and iP treatment did not increase the resistance of rice against Magnaporthe oryzae13. This could explain why increased cZ or iP levels in some samples after treatment with the CK-deficient mutants had no effect on resistance against Pto and emphasizes the role of tZ levels as a key parameter in G20-18-mediated biocontrol.

Table 1 Cytokinin levels in Arabidopsis Col-0 48 h post infiltration with Pfl strains. Full size table

The analyses of a large set of individual CK levels revealed subtle though distinct changes in the host plant. In both datasets specific differences between G20-18 and its derivatives were successfully monitored and appeared to be robust even against variable background levels indicated by the variable CK levels in the control samples (Supplementary Table 1 and Supplementary Table 2). The complex regulation of CK levels in Arabidopsis depends on nine biosynthesis and seven catabolism genes that are potentially affected by Pfl, similar to other Arabidopsis-microbe interactions (eFP browser33), which could be responsible for the different CK ratios between G20-18-treated and control samples in the two sets in addition to microbial CK production (Supplementary Table 1 and Supplementary Table 2). In addition, the infiltration process, ambient conditions, inter-conversions and transport of CKs contribute to complex spatiotemporal dynamics at the cellular level, which are difficult to resolve by CK determination in plant tissue. Considering the known activity of CKs at low concentrations, the subtle differences caused by bacterial CK production linked to the CK-mediated plant defence ensures minimal interference with general plant CK homeostasis and thus minimal perturbation of other plant processes.

G20-18 biocontrol limits pathogen growth

The differential efficacy in biocontrol by G20-18 and its CK-deficient mutants could result from growth variations of the Pfl strains in planta, since the number of living Pfl cells may determine biocontrol by competition with Pto for nutrients and space34,35. Such growth differences could depend on their capacity to produce CKs, which may interfere with bacterial quorum sensing36 or the communication between microbe and plant for successful niche establishment37. Additionally, growth defects could be caused by pleiotropic effects of the applied mutagenesis unrelated to the CK deficiency, while reduced viability of the Pfl mutants could cause lower CK production. Therefore, the number of viable Pfl cells in planta at the time-point of Pto infection - 48 hours post infiltration (hpi) of Pfl -, was determined for G20-18 and the different mutant strains (Fig. 4a). Similar numbers of viable cells were determined directly after the infiltration (0 hpi) and at 48 hpi for all strains except CNT2 for which viable cells decreased (significantly compared to CNT1). Based on these data, growth differences between G20-18 and the analysed mutant strains can be excluded as the cause of the variations in their biocontrol abilities.

Figure 4 Pfl G20-18 and its CK-deficient mutants do not differ in growth, but differentially affect Pto proliferation in planta. (a) Number of viable Pfl cells in Arabidopsis leaves 0 hours post infiltration (hpi) with 107 cfu ml−1 and at the time-point of Pto infection (48 hpi). n = 27. (b) Number of viable Pto cells harbouring pMP4662 in Arabidopsis leaves directly after infiltration (0 hpi) with 105 cfu ml−1 and at 72 hpi. n = 18. Data are means ± s.e., letters indicate different significance groups (P < 0.05). Full size image

As CKs can directly contribute to a favourable physiological status by modulating primary metabolism8,9 and thus potentially affect tissue integrity, suppression of symptom development during CK-mediated resistance does not necessarily correlate with restriction of pathogen growth28, which is a direct result of increased resistance. To discriminate between increased resistance induced by G20-18-derived CKs and general impact on tissue integrity, we determined Pto growth in planta after pre-treatment with G20-18 and its CK-deficient mutants. Pto proliferation was significantly reduced after G20-18 pre-treatment compared to the mutant and mock pre-treatments at 72 hpi (Fig. 4b) and thus restricted Pto proliferation can be considered as the cause for reduced symptom development in the leaf infiltration assays. Further, Pto proliferation was strongly negatively correlated with the tZ levels determined at the time-point of infection (Table 1) following pre-treatments with the different Pfl strains (ranked data, Spearman’s correlation coefficient of −0.8). G20-18 pre-treatment resulted in the lowest Pto proliferation and the highest tZ levels, followed by pre-treatments with the CNT transposon mutants which similarly caused lower tZ levels and higher Pto proliferation comparable to mock treatment, while ΔmiaA pre-treatment resulted in the lowest tZ levels and the highest Pto proliferation. This correlation supports the role of specific active CKs in determining biocontrol activities by inducing defence responses that act directly on the pathogen in a dose-dependent manner, similar to resistance effects induced by exogenously applied CKs11,12, which in a certain range can act in a dose-dependent manner and require specific threshold levels to be active.

G20-18 biocontrol requires plant pathways

Pfl G20-18 showed suppressive effects on Pto symptom development and multiplication in Arabidopsis indicating direct activation of plant defences, which were lacking after pre-treatment with CK-deficient Pfl mutants. To dissect the underlying plant mechanisms, the efficiency of G20-18-mediated biocontrol was determined in several Arabidopsis lines impaired in phytohormone and/or defence-related mechanisms (Fig. 5a). Since we identified microbial CK production as a determinant of Pfl G20-18-mediated biocontrol against Pto, we assumed functional CK perception as the initial step of CK signalling in the plant to be essential. In Arabidopsis CK perception depends on the three membrane-bound histidine kinases AHK2, AHK3 and AHK4/CRE1/WOL6. The function of these Arabidopsis CK receptors in G20-18 biocontrol was assessed in the double mutant lines ahk2-2/ahk3-3 and cre1-12/ahk3-3 and the triple mutant cre1-12/ahk2-2/ahk3-3(+/−) (homozygous for cre1-12 and ahk2-2, heterozygous for ahk3-3)38. G20-18-mediated biocontrol was reduced in all three mutant lines (Fig. 5a), illustrated by significantly elevated Pto symptom development compared to the wild-type Col-0 (Fig. 5b). This indicates that all three receptors function as signalling components of CK-dependent biocontrol by G20-18, which is supported by the finding that the triple mutant exhibited the strongest defect (Fig. 5b). However, a rudimentary G20-18 biocontrol effect is still observed in these mutant plant lines, which is either due to residual CK perception or is unrelated to G20-18 CK production and/or plant CK signalling.

Figure 5 Pfl G20-18 biocontrol depends on functional hormonal and defence pathways of the host. (a) Pto symptom development (right leaf halves) 4 days post infection (dpi) with 106 cfu ml−1 in indicated Arabidopsis lines after Pfl G20-18 pre-treatment. (b) Average Pto symptom scores 4 dpi with 106 cfu ml−1 in indicated Arabidopsis mutant or transgenic lines (red bars) compared to Col-0 (Col-gl for myc2) wild-type (blue bars) pre-treated with Pfl G20-18 or the appropriate mock. Data are means ± s. e. n ≥ 28, letters indicate different significance groups (P < 0.05). Full size image

SA was demonstrated as a key central defence signalling component of CK-mediated resistance, mainly depending on NPR1 signalling, against Pto in Arabidopsis10, but also as a parameter of CK-induced resistance or defence responses in other plant species12,13,28. The role of SA in G20-18-mediated biocontrol was assessed in Arabidopsis lines either overexpressing nahG (35S::nahG), a SA-degrading enzyme from Pseudomonas putida39, or defective in SA biosynthesis (sid2)40 or SA signalling (npr1)41. In agreement with the known SA-dependent tZ-mediated resistance effect in Arabidopsis10, G20-18 pre-treatment was almost completely ineffective in these lines as Pto symptoms were not suppressed (Fig. 5a). 35S::nahG, sid2 and npr1 (Fig. 5b) showed Pto symptoms after G20-18 treatment comparable to the mock treatment in the plant mutants and Col-0 wild-type, hence SA accumulation as well as functional SA signalling have to be considered as major parameters in CK-mediated biocontrol.

To examine involvement of the defence-related phytohormones JA and ET, which are important for inducing ISR as part of biocontrol and for priming effects mediated by beneficial microbes4,20, G20-18 biocontrol assays were performed in the mutant lines myc2 (jin1)42 which is partially insensitive to JA43 and ein244, which is insensitive to ET. In both Arabidopsis lines, the suppressive effect of G20-18 on Pto symptoms was reduced (Fig. 5a). Although this reduction was significant compared to Arabidopsis wild-type Col-gl (myc2) and Col-0 (ein2; Fig. 5b), it is considerably lower than observed in SA-related plant mutants, indicating a minor role of JA and ET in this biocontrol mechanism in leaf infiltration assays. As phytoalexins also potentially contribute to biocontrol effects as antimicrobial compounds45,46, G20-18 biocontrol effects were analysed in Arabidopsis pad347 and cyp7948 mutants deficient in camalexin, the key phytoalexin in Arabidopsis49. In both lines, the effect of G20-18 on Pto was reduced, as evidenced by stronger symptom development compared to Col-0 (Fig. 5a), which was significantly lower, however, than the mock controls (Fig. 5b). These data suggest a minor role of camalexin in G20-18-mediated biocontrol in the leaf infiltration assays, which possibly depends on microbial CKs similar to CK-induced resistance effects shown in tobacco12,14,15.