Modulating plant primary amino acid metabolism as a necrotrophic virulence strategy

Abstract

The fungal plant pathogen Botrytis cinerea is the causal agent of the “gray mold” disease on a broad range of hosts. As an archetypal necrotroph, B. cinerea has evolved multiple virulence strategies for inducing cell death in its host. Moreover, progress of B. cinerea colonization is commonly associated with induction of senescence in the host tissue, even in non-invaded regions. In a recent study, we showed that abscisic acid deficiency in the sitiens tomato mutant culminates in an anti-senescence defense mechanism which effectively contributes to resistance against B. cinerea infection. Conversely, in susceptible wild-type tomato a strong induction of senescence could be observed following B. cinerea infection. Building upon this earlier work, we here discuss the immune-regulatory role of a key senescence-associated protein, asparagine synthetase. We found that infection of wild-type tomato leads to a strong transcriptional upregulation of asparagine synthetase, followed by a severe depletion of asparagine titers. In contrast, resistant sitiens plants displayed a strong induction of asparagine throughout the course of infection. We hypothesize that rapid activation of asparagine synthetase in susceptible tomato may play a dual role in promoting Botrytis cinerea pathogenesis by providing a rich source of N for the pathogen, on the one hand, and facilitating pathogen-induced host senescence, on the other.

Results and Discussion

The amino acid asparagine is one of the major metabolic products in senescing leaves in plants.¹ Asparagine has the highest N:C ratio (2:4) among all amino acids, which makes it an efficient metabolite for nitrogen transport during the process of senescence.^2,3 The major route of asparagine synthesis is governed by the ATP-dependent transfer of the amino group of glutamine to a molecule of aspartate to form glutamate and asparagine, a reaction catalyzed by the enzyme asparagine synthetase (AS).³ AS is also known as a key senescence associated gene (SAG),⁴ with possible roles in plant-pathogen interactions.^5,6 It has been proposed that AS is involved in metabolic alterations that facilitate host cell death during plant-pathogen interactions; hence promoting resistance against biotrophic pathogens. However, such metabolic alterations may also render susceptibility to necrotrophic infections.⁷ For instance, an early (5 hpi) induction of AS1 in pepper was shown to be involved in resistance against the hemibiotrophic bacterial pathogen Xanthomonas campestris pv vesicatoria, while later (15–20 hpi) induction of the enzyme (during the necrotrophic phase of the pathogen infection) was associated with susceptibility.⁸

Taking advantage of the abscisic acid (ABA)-deficient sitiens mutant of tomato, which displays high levels of immunity against the necrotrophic fungus Botrytis cinerea (B. cinerea),^9-11 our research aims to decipher the molecular underpinnings of necrotroph resistance in plants. In our latest study, we showed that the observed resistance response in sitiens is dependent on timely restructuring of the central C/N metabolism, particularly the GABA-shunt.¹² The GABA shunt is a cytosolic-mitochondrial pathway that connects amino acid metabolism to the tricarboxylic acid (TCA) cycle, with possible roles in plant defense mechanisms against phytopathogens.⁷ According to our proposed model, concurrent hyper-activation of the cytosolic glutamine synthetase (GS1) and the GABA-shunt in sitiens are involved in maintaining basic metabolism in the challenged tissue to tightly control the extent and localization of cell death.¹² As a result, the extent of senescence is spatiotemporally more restricted in the resistant sitiens mutant compared with susceptible wild-type tomato plants¹² (Fig. 1). In agreement with this, we also found lower levels of total protein degradation and reduced activation of the SAG, glutamate dehydrogenase (GDH), in sitiens vs. wild-type seedlings.

Figure 1. Induction of senescence in non-inoculated areas (red arrows) of an infected wild-type (WT) leaf, compared with an inoculated leaf of the sitiens (Sit) mutant at 72 hpi. Fifth or sixth leaves of 5-wk-old plants (7 leaf stage) were inoculated according to Curvers et al. (2010)¹¹ by carefully placing 4 droplets (per leaflet) containing 10-µL of conidial suspension (5 × 10⁵ spores mL⁻¹ in 0.01 M KH₂PO₄ and 6.67 mM Glucose) on the adaxial leaf surface. Inoculated leaves were placed in enclosed trays to retain high relative humidity and incubated at 22 °C under continuous dark conditions.

Building upon these earlier findings, we sought to extend our analysis by monitoring the expression of another key SAG, asparagine synthetase 1 (AS1; GeneBank accession number AY240926.1). As shown in Figure 2, AS1 showed a tremendous upregulation in the wild-type tomato in response to B. cinerea infection, whereas such strong induction was not seen in the sitiens mutant (Fig. 2). Figure 3 indicates that the levels of asparagine increased in all mock-treated plants, showing the effect of dark incubation on asparagine accumulation, as suggested previously.¹³ Moreover, asparagine titers in B. cinerea-inoculated sitiens plants increased significantly during the course of infection (Fig. 3), whereas infected wild-type seedlings showed a remarkable drop in asparagine content (Fig. 3). Particularly, the highest level of depletion of asparagine can be seen at 48 hpi in the wild-type plant, while such a severe depletion did not occur for other amino acids (Fig. 4). Finally, an in vitro assay revealed that asparagine is a perfect nitrogen (N) source for B. cinerea, resulting in rapid mycelial growth and dense fungal sporulation (Fig. 5).

Figure 2. Expression of asparagine synthetase in wild type (WT) and sitiens (Sit) plants following infection with B. cinerea (M: mock; I: infected). The qPCR analysis was performed following the protocol described previously¹² using AS1-specific primers; Forward: TCCCCTTTGG TGTTCTGCTC TCG, Reverse: TGCTCCCCAT TGCTTAGCAG C. Error bars represent ± SE of 3 biological replicates.

Figure 3. Alterations in asparagine levels in mock-treated (M) and Botrytis-inoculated (I) sitiens (Sit) and wild-type (WT) tomato plants at different time points post inoculation. Metabolic analysis of free amino acid levels was conducted as described before.¹² Error bars represent ± SE of 3 biological replicates.

Figure 4. Amino acid depletion ratios (levels in mock vs. infected samples) in Botrytis-infected sitiens (Sit) and wild-type (WT) tomato at 48 hpi. Among all amino acids, asparagine displays the most marked depletion in the wild-type plant.

Figure 5. In vitro effect of asparagine (Asn, 5mM) on the growth and sporulation of B. cinerea grown on Gamborg B5 salt base medium (B5 base: with no carbon and nitrogen sources). Sucrose (S) was used as carbon source. The black circles indicate the place of the inoculation plaque.

Induction of senescence in the host is a well-studied pathogenicity mode for the necrotrophic pathogen B. cinerea.¹⁴ Accordingly, it appears that in wild-type tomato susceptibility to B. cinerea is strongly correlated with induction of classic SAGs, including AS. AS has also been shown to be an ABA-responsive gene that is upregulated in response to different abiotic stresses.^15,16 This might explain why in the ABA-deficient sitiens mutant, compared with the wild-type plant, the AS gene is only slightly induced upon B. cinerea infection. Moreover, the relatively low level of AS activation in sitiens might consequently prevent the cell N reservoir (glutamine and aspartate) from being converted to transportable forms of amino acid (asparagine), thus providing more cytosolic supply for the GABA-shunt-mediated, anti-senescence defense response seen in the mutant.¹²

Previous studies revealed that, depletion in sugar levels in tomato plants subjected to prolonged dark conditions also result in strong activation of the AS gene.¹⁷ Therefore, the strong induction of AS in B. cinerea-infected wild-type tomato (Fig. 2) could be explained through both catabolization of the host apoplastic sugar content by the pathogen, and continuous incubation of tomato leaves in darkness. Interestingly, B. cinerea-induced upregulation of AS in the wild-type plant seems to be followed by strong depletion of asparagine content in the infected leaves, suggesting a causal link between both phenomena. Having the highest N content among all amino acids, asparagine may serve as a rich N source for B. cinerea in vitro (Fig. 5), and possibly also in planta. In view of these findings, it is tempting to speculate that strong induction of AS in susceptible tomato plants plays a dual role in the pathogenicity of B. cinerea by both facilitating pathogen-initiated host senescence and providing a rich N reservoir to support fungal growth in planta. Future studies should be focused on exploring the molecular mechanism(s) by which B. cinerea targets AS and manipulates the plant’s primary metabolism for its own benefit.

Glossary

Abbreviations:

AS

asparagine synthetase

GDH

glutamate dehydrogenase

SAG

senescence-associated gene

hpi

hours post inoculation