Biological control using beneficial microbes is receiving considerable attention for managing plant diseases in a wide range of crops, thus reducing chemical pesticides input. In fact, according to the Farm to Fork Strategy, part of the European Union Green Deal (European Commission, 2020), limitations on chemical pesticides foster the importance for innovative and sustainable solutions in view of consumer acceptance for chemical-free food.
Recent studies reveal that Streptomyces are excellent BCAs against various plant pathogens by employing different mechanisms of action (Vurukonda et al. 2018). For instance, they can suppress phytopathogenic microbial targets by producing specific bioactive molecules (Khan et al. 2023). Apart from antibiosis, Streptomyces were shown to trigger localised and systemic resistance in plants against a broad range of pathogens and insect herbivores (Pieterse et al. 2014), and colonisation of specific plant “sites” is crucial in their ecological fitness to put in place successful mechanisms of action (Pieterse et al. 2014). Streptomyces are known as major bacterial colonisers of plant roots and important microbial components of disease-suppressive soils; however, they rarely colonise plant leaves. Thus, their potential to protect crops from aerial pathogens has been poorly investigated (Vergnes et al. 2020).
In the current study, the Streptomyces sp. DLS2013 showed the capability to epiphytically colonise tomato phyllosphere after foliar application, by establishing and maintaining constant its population over the time, with a magnitude order of approx. 104 CFU per gr of leaf tissue up to seven days. On the contrary, its capability to endophytically colonise leaves was not recorded (Table 2). Therefore, these results demonstrate that DLS2013 can also dwell on plant leaves, extending its potential use to protect plants against foliar diseases as shown in other experiments (Vergnes et al. 2020).
RNA-Seq differential gene expression analysis was performed to investigate possible transcriptional changes induced by DLS2013-plant interaction over time, and to elucidate the underlying biological processes involved in the induced defence response in treated tomato plants. The GO and pathway enrichment analysis indicated that DLS2013 positively affected the upregulation of functionally annotated DEGs in seven BP categories related to the defence response at all three time points (i.e., T1, T2 and T3) (Fig. 4), and that 67 upregulated genes in DLS2013-treated plants pertain to PA-Phen, JA, and SA metabolic pathways. On the other end, several genes, involved in photosynthesis and flavonoid biosynthesis, were downregulated. In details, we observed the early up-regulation of five genes involved in the biosynthesis of PAs (i.e., ARG, ADC, ODC, SAMDC and SPS), which may elicit the synthesis of the major polyamines’ putrescine, spermidine, and spermine (Fig. 6; left). These PAs are effective defence responses to (a)biotic stresses by actively contributing to (i) potentiation of the ROS burst, (ii) induction of defence-related genes, or through (iii) crosstalk with hormonal pathways, such as those of ethylene or SA (Seifi and Shelp, 2019).
Notably, during the host-pathogen co-evolution, Pseudomonas syringae has developed the ability to secrete multiple phytotoxins that can inhibit the biosynthesis of PAs (i.e., phaseolotoxin and mangotoxin), or their ability to trigger the immune oxidative burst (i.e., phevamine A). This finding supports the pathogen's need to counteract or suppress the defence responses elicited by PAs (Fig. 6) (Gerlin et al. 2021).
Furthermore, five overexpressed genes involved in the Phen-biosynthesis, such as: 4CL, HCT, C3’H, CAMT1 were identified (Fig. 6; right). Stilbenoids and phenylpropanoids, produced during the initial stages of the lignin biosynthesis, are specialised metabolites with direct harmful effect against microbial pathogens or insect targets and with additional protective functions of plant tissues from toxic effect of ROS, contributing to biotic resistance in plants (Ramaroson et al. 2022; Valletta et al. 2021).
Additional pathways, whereby DLS2013 introduced the up-regulation of genes, were potentially associated to the JA-mediated defence responses (Fig. 7). Here, we identified upregulated genes controlling the chloroplast (i.e., PLA, LOX, AOS and AOC) and peroxisome (i.e., OPR3) phases of JA biosynthesis and the production of its volatile derivative, Jasmonic Acid Methyl Ester (Me-JA), in the cytoplasm (i.e., JA-associated MYC2-like (JAM)). JAM proteins are responsible for the methylation of JA to form its bioactive metabolites (Seo et al. 2001). Me-JA enhanced defence response in plants through different mechanisms: (i) activating the expression of defence genes; (ii) inducing accumulations of defence compounds (e.g., phenolics); (iii) regulating photosynthesis, such as stomatal closure (Yu et al. 2019). Among the JA-signalling genes, several up-regulated DEGs were identified (Fig. 7), suggesting potential induction of the JA-mediated defence response, known to play a key role against chewing insects and necrotrophic pathogens (Smith et al. 2009). At the same time, we have identified five different up-regulated JA zinc-finger inflorescence meristem (ZIM)-domain (JAZ) genes (Supplementary Table 3), which are integral components of the JA signalling. JAZs are negative regulators of JA-induced genes and are tagged by the SCFCOI1 complex for the 26S proteasome-mediated degradation induced by the nuclear translocation of JA-Ile (Gupta et al. 2020). Studies conducted in Arabidopsis thaliana showed that Pst DC 3000 might trigger the activation of JA-dependent defence responses by producing coronatine—a JA-Ile mimic (Zheng et al. 2012). This results in a hormonal imbalance and activation of inappropriate defence responses by suppressing SA-dependent defence responses and prevention of stomatal closure, which facilitates bacterial entry into the leaf, with the promotion of disease symptoms (Melotto et al. 2006). At the up-regulation of the JA-mediated defence response corresponded the increased expression of PR-3 (chitinase), PR-6 (proteinase inhibitors), PR-9 (peroxidases) and PR-10 (ribonucleases) biosynthesis pathways (Supplementary Table 3), which mainly act against necrotrophic pathogens and insects (Yu et al. 2019). PR-3 proteins can catalyse the cell wall degradation of microbial pathogens and insects and induce a hypersensitivity response to prevent the pathogen's spread to other tissues (Ali et al. 2020). Among PR-6 proteins, the serine-type endopeptidase inhibitors (PIs) may act by reducing the ability of the attacker to (i) use its lytic enzymes necessary for fungal pathogenicity; (ii) complete viruses replication cycles (Sels et al. 2008). On the other hand, tyrosine inhibitors (TIs) can act against the digestive proteases (e.g., trypsin) used by herbivorous insect pests and contribute to the inhibition of phytopathogenic fungi (Rodríguez-Sifuentes et al. 2020). For PR-9 and PR-10 biosynthesis pathways, we identified seven (e.g., CAD and POXs) and three (i.e., T2-ribonucleases) up-regulated DEGs, respectively. PR-9 is a specific type of peroxidase that acts in cell wall ramification by catalysing lignification, making it difficult for the pathogen to cross the barrier (dos Santos et al. 2023). PR-9 also enhances resistance against pathogens by producing ROS and maintaining plant cells' redox homeostasis (Sellami et al. 2022). PR-10 consists of small acidic proteins, involved in the accumulation of ROS, antifungal, antimicrobial action and activity of RNase and DNase (Lopes et al. 2023).
The up-regulation of JA and its derivative MeJA, has been reported to modulate the accumulation of terpenes, as well (Yu et al. 2019). In the present study, we found the up-regulation of seven DEGs involved in terpene biosynthesis (Supplementary Table 3), which can protect plants against herbivory and attacks from microbial pathogens and invertebrate pests (Celedon and Bohlmann, 2019). Moreover, terpenes can also act as plant-to-plant signalling: recent studies revealed that volatile terpenes could alter distinct internal signalling routes leading to defence responses against pathogens (i.e., induced systemic resistance (ISR) and systemic acquired resistance (SAR)) in receiver plants (Frank et al. 2021; Rosenkranz et al. 2021). JA pathways often operate synergistically with the ET-signalling. Indeed, our results also showed an up-regulation of genes involved both in ethylene- biosynthesis (i.e., ACS and AOS) and signalling (i.e., ERF1) (Supplementary Table 3). ERF transcription factors play a critical role in plant immunity, integrating ethylene and signals to fine-tune the defence response to different necrotrophic pathogens (Lorenzo et al. 2003).
Besides the PA-Phen metabolism and JA/ET defence responses, different up-regulated DEGs were found among the SA-mediated defence responses (Fig. 8). These DEGs were found both in plastid (such as 4CL, PBS3s, and EPS1s) and nucleus (specifically NPR1). NPR1 is a protein that acts as a master regulator of plant SA-signalling, playing a crucial role in plant immunity by triggering the activation of PR-1 and PR-5 and by suppressing the JA signalling pathway (Chen et al. 2019). Accordingly, our results showed the up-regulation of three and two DEGS for PR-1 and PR-5 genes, respectively. The biochemical functions of PR-1 proteins have yet to be completely understood, even though most PR-1 orthologs of different plant species exhibit in- vitro antimicrobial activity. However, a recent study revealed that tobacco PR-1a exhibits sterol binding capabilities, thereby preventing pathogen growth by sequestering essential sterols away from the invading organism (Gamir et al. 2017). PR-5 encodes a thaumatin-like protein, rated as the potent antifungal proteins in plants (Ali et al. 2018). Accordingly, overexpression of Solyc08g080660, one of the PR-5 genes upregulated in this study, provides resistance to five soil-borne diseases caused by fungal pathogens in tomato plants (Li et al. 2023). The PR-1 and PR-5 genes are markers of SA signalling pathway (Guo et al. 2023), pivotal in mounting plant defence mechanisms against biotrophic and hemi-biotrophic pathogens by initiating SAR (Bari and Jones, 2009). Conversely, the results of the GO and pathway enrichment analysis showed a significant downshift in gene expression related to other primary metabolic activities, such as photosynthesis and flavonoid-associated pathways, at T1 and/or T2 time points (Fig. 4; additional information in Supplementary Table 3). The suppression of photosynthesis-linked and carbohydrate metabolic pathways is seen as a strategic defensive move by plants. This enables them to (i) re-channel nitrogen resources without severely compromising their short-term photosynthetic output (Bilgin et al. 2010), and (ii) continue utilizing carbon for the essential biosynthesis of defence signals under conditions of lowered carbon supply (Smith and Stitt, 2007). Indeed, findings that photosynthesis, sugar metabolism, and ROS detoxification genes share regulatory elements in their promoter regions indicate that the transcriptional shifts in bolstered defence responses are meticulously synchronized (Berger et al. 2007). The decrease in transcription of genes responsible for producing flavonoids and the increased expression of genes involved in lignin production suggested that the common precursor p-coumaroyl CoA was mainly addressed toward lignin rather than flavonoid biosynthesis. This adaptive response was correlated with increased resistance to the hemibiotrophic pathogen Pseudomonas syringae pv. actinidiae in kiwifruit (Li et al. 2021). Overall, the differential gene expression analysis by RNA-Seq highlighted the capability of DLS2013 to trigger defence response by priming PA-Phen, JA, and SA pathways without pathogen infections. This leads to improved protection against a wide range of potential plant pathogens with minimal impact on fitness, compared with independent activation of each pathway (Van Wees et al. 2008). Nevertheless, it is commonly accepted that the most predominant plant resistance against necrotrophic or hemibiotrophic pathogens depends on the antagonism between JA-dependent defence responses and SA-dependent defence signalling (Gupta et al. 2020). When plants are primed with BCAs, their ability to withstand pathogen attacks hinges on fine-tuning their pre-activated immune responses and the pathogen's attempts to counteract these defences.
In the in vivo assay, tomato plants pre-treated with DLS2013 showed a reduction of bacterial speck DI by 67%, with respect to the control (Mock) (Fig. 2). On the contrary, DLS2013 did not show antimicrobial activity in vitro against Pst (Fig. 1). This negative correlation observed between Pst-growth inhibition (in vitro assay) and Pst-DI (in vivo assay) indicates that plant resistance to hemibiotrophic phytobacteria relies with the activation of plant defence responses Therefore, we have further investigated the ability of DLS2013 to elicit resistance in tomato plants against the hemibiotrophic pathogen Pst, by assessing the expression of six genes associated with the following distinct defence response pathways: (i) phenolics (HCT gene), (ii) polyamines (ARG gene), (iii) JA (LOX 4, JAZ 25 and PR-3 genes) and (iv) SA (PR-1 gene) at 24 hours-post challenging, as possible markers of induced defence.
RT-qPCR analysis revealed an increased HCT transcript in Pst inoculated tomato plants, that could be attributed to the antimicrobial property of phenolics against pathogens (López-Gresa et al. 2011). Compared to Pst inoculation, a similar level of HCT expression was observed in DLS2013-treated plants, highlighting the potential role of DLS2013 in tomato defence priming. Although the HCT transcription was induced when this BCA was applied alone, it was significantly lower in primed-plants inoculated with Pst. This lower level of HCT transcription in DLS2013 + Pst treated plants could be related to the transcript’s abundance during the tripartite interaction among plant-Streptomyces sp.-pathogen, as already found by Abbassi et al. (2019) for other defence related genes (e.g., tomato peroxidase (TPX1)). A similar expression profile was displayed by ARG gene, where Pst inoculated plants showed the highest transcription level. Intriguingly, in primed plants then challenged with Pst, ARG expression was the same of control (Mock). Here, the effect of Pst DC 3000 phytotoxins (i.e., phaseolotoxin) might have additively contributed to the downregulation of PAs biosynthesis. Nevertheless, overexpression of both HCT and ARG genes in plants challenged with Pst or DLS2013 alone, may indicate that plants try to produce additional antimicrobial compounds against Pst or as a response to BCA priming (Vogt, 2010; Dixon, 2001), and protect themselves from the H2O2 generated in the early response to biotic challenges (Gerlin et al. 2021). Beyond, Pseudomonas syringae pathogens have evolved various defence strategies, one of which includes multidrug resistance (MDR) efflux pumps. These pumps help the pathogens to counteract the potentially toxic compounds of microbial antibiotics, and plants’ antimicrobial compounds (Santamaría-Hernando et al. 2019), as observed in both in vitro and in vivo experiments of this study. No significant differences were found between treatments and control (Mock) for the LOX 4 and PR-3 target genes, which were chosen as markers for the biosynthesis and signalling of the JA defence response. During the first experiment (i.e., RNA-Seq sequencing), the overexpression of LOX 4 up to 24 hours after DLS2013-treatment was associated with comparable up-regulation of PR-3 genes for the JA signalling (Supplementary Table 3). It is noteworthy, the declined expression of genes responsive to JA (including LOX 4) in primed plants after Pst inoculation, was already reported in similar studies (Elsharkawy et al. 2021). This decline in the expression of the JA pathways can be explained by the transcript’s abundance of both LOX and PR-3 during plant-Streptomyces sp. interaction, as already detected for HCT and ARG genes. Furthermore, the expression level JAZ 25 gene was significantly increased in response to the DLS2013-treatments alone or in primed plants than challenged with Pst, compared to control plants (Mock) and un-primed plants challenged with Pst. This finding indicates the key role of JAZ repressors as important mediators of early basal and subsequent secondary plant defence response, confirming previous studies on the mutually function of JAZ genes to reduce Pseudomonas syringae symptoms (Gupta et al. 2020; Toum et al. 2016). Notably, Pst DC3000 interacts in plant cells with phytotoxic virulence factor coronatine (COR), which led to the degradation of JAZs, who in turn interferes with hormone signalling by causing the upregulation of JA-responsive genes, inhibiting SA-dependent responses, and resulting in an enhanced plant susceptibility (Cui et al. 2010). Our results confirmed these findings, indeed the function of overexpressed JAZ genes in both primed unchallenged and challenged with Pst tomato plants compared to controls was positively correlated with significant increases of PR-1, marker gene for SA-dependent defence signalling, that enhances plant resistance to infection by hemibiotrophic pathogens, such as Pst (Zhang et al. 2022).
In this study, we demonstrate that DLS2013 can colonize tomato leaves as an epiphyte and activate the plant's defence system at the transcriptional level through various molecular pathways, including SA, JA, PAs, and Phen biosynthesis, as summarised in Figs. 6, 7, and 8. Moreover, the negative correlation between Pst- growth inhibition (in vitro assay) and protection against bacterial speck (in vivo assay) of primed tomato plants confirmed the activation of effective defence responses, mainly related with the overexpression of SA-dependent defence signalling and the suppression of the JA dependent defence response, where JAZ 25 gene may play a negative regulatory key role in tomato resistance to bacterial speck. Consistent with recent investigations on the JAZ 25 gene and its central role in developing pathogen resistance in tomatoes (Sun et al. 2021), our results propose this specific gene as additional benchmark for defence priming. However, further research is needed to unveil the potential of DLS2013 to boosts plant disease resistance to a broader range of plant pathogens by activating multiple defence pathways, such as SA, JA, PA-Phen biosynthesis, and assessing its beneficial protection against necrotrophic pathogens and insects. Overall, this study might be a valuable resource to further investigate associations between DLS2013 and different pests, aiming to foster sustainable biological control methods for the integrated pest management in tomato.