The phenylalanine ammonia-lyase inhibitor AIP induces rice defence against the root-knot nematode Meloidogyne graminicola.

The phenylalanine ammonia-lyase (PAL) enzyme catalyses the conversion of l-phenylalanine to trans-cinnamic acid. This conversion is the first step in phenylpropanoid biosynthesis in plants. The phenylpropanoid pathway produces diverse plant metabolites that play essential roles in various processes, including structural support and defence. Previous studies have shown that mutation of the PAL genes enhances disease susceptibility. Here, we investigated the functions of the rice PAL genes using 2-aminoindan-2-phosphonic acid (AIP), a strong competitive inhibitor of PAL enzymes. We show that the application of AIP can significantly reduce the PAL activity of rice crude protein extracts in vitro. However, when AIP was applied to intact rice plants, it reduced infection of the root-knot nematode Meloidogyne graminicola. RNA-seq showed that AIP treatment resulted in a rapid but transient upregulation of defence-related genes in roots. Moreover, targeted metabolomics demonstrated higher levels of jasmonates and antimicrobial flavonoids and diterpenoids accumulating after AIP treatment. Furthermore, chemical inhibition of the jasmonate pathway abolished the effect of AIP on nematode infection. Our results show that disturbance of the phenylpropanoid pathway by the PAL inhibitor AIP induces defence in rice against M. graminicola by activating jasmonate-mediated defence.

Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

The diversity of salicylic acid biosynthesis and defense signaling in plants: Knowledge gaps and future opportunities.

The phytohormone salicylic acid (SA) is known to regulate plant immunity against pathogens. Plants synthesize SA via the isochorismate synthase (ICS) pathway or the phenylalanine ammonia-lyase (PAL) pathway. The ICS pathway has been fully characterized using Arabidopsis thaliana, a model plant that exhibits pathogen-inducible SA accumulation. Many species including Populus (poplar) depend instead on the partially understood PAL pathway for constitutive as well as pathogen-stimulated SA synthesis. Diversity of SA-mediated defense is also evident in SA accumulation, redox regulation, and interplay with other hormones like jasmonic acid. This review highlights the contrast between Arabidopsis and poplar, discusses potential drivers of SA diversity in plant defenses, and offers future research directions.

Constitutive expression of JASMONATE RESISTANT 1 induces molecular changes that prime the plants to better withstand drought.

In this study, we investigated Arabidopsis thaliana plants with altered levels of the enzyme JASMONATE RESISTANT 1 (JAR1), which converts jasmonic acid (JA) to jasmonoyl-l-isoleucine (JA-Ile). Analysis of a newly generated overexpression line (35S::JAR1) revealed that constitutively increased JA-Ile production in 35S::JAR1 alters plant development, resulting in stunted growth and delayed flowering. Under drought-stress conditions, 35S::JAR1 plants showed reduced wilting and recovered better from desiccation than the wild type. By contrast, jar1-11 plants with a strong reduction in JA-Ile content were hypersensitive to drought. RNA-sequencing analysis and hormonal profiling of plants under normal and drought conditions provided insights into the molecular reprogramming caused by the alteration in JA-Ile content. Especially 35S::JAR1 plants displayed changes in expression of developmental genes related to growth and flowering. Further transcriptional differences pertained to drought-related adaptive systems, including stomatal density and aperture, but also reactive oxygen species production and detoxification. Analysis of wild type and jar1-11 plants carrying the roGFP-Orp1 sensor support a role of JA-Ile in the alleviation of methyl viologen-induced H2 O2 production. Our data substantiate a role of JA-Ile in abiotic stress response and suggest that JAR1-mediated increase in JA-Ile content primes Arabidopsis towards improved drought stress tolerance.

Lack of antagonism between salicylic acid and jasmonate signalling pathways in poplar.

Salicylic acid (SA) and jasmonic acid (JA) often play distinct roles in plant defence against pathogens. Research from Arabidopsis thaliana has established that SA- and JA-mediated defences are more effective against biotrophs and necrotrophs, respectively. These two hormones often interact antagonistically in response to particular attackers, with the induction of one leading to suppression of the other. Here, we report a contrasting pattern in the woody perennial Populus: positive SA-JA interplay. Using genetically engineered high SA lines of black poplar and wild-type lines after exogenous hormone application, we quantified SA and JA metabolites, signalling gene transcripts, antifungal flavonoids and resistance to rust (Melampsora larici-populina). Salicylic acid and JA metabolites were induced concurrently upon rust infection in poplar genotypes with varying resistance levels. Analysis of SA-hyperaccumulating transgenic poplar lines showed increased jasmonate levels, elevated flavonoid content and enhanced rust resistance, but no discernible reduction in growth. Exogenous application of either SA or JA triggered the accumulation of the other hormone. Expression of pathogenesis-related (PR) genes, frequently used as markers for SA signalling, was not correlated with SA content, but rather activated in proportion to pathogen infection. We conclude that SA and JA pathways interact positively in poplar resulting in the accumulation of flavonoid phytoalexins.

Biological and biorational management of blast diseases in cereals caused by Magnaporthe oryzae.

Blast diseases, caused by the fungal pathogen Magnaporthe oryzae, are among the most destructive diseases that occur on at least 50 species of grasses, including cultivated cereals wheat, and rice. Although fungicidal control of blast diseases has widely been researched, development of resistance of the pathogen against commercially available products makes this approach unreliable. Novel approaches such as the application of biopesticides against the blast fungus are needed for sustainable management of this economically important disease. Antagonistic microorganisms, such as fungi and probiotic bacteria from diverse taxonomic genera were found to suppress blast fungi both in vitro and in vivo. Various classes of secondary metabolites, such as alkaloids, phenolics, and terpenoids of plant and microbial origin significantly inhibit fungal growth and may also be effective in managing blast diseases. Common modes of action of microbial biocontrol agents include: antibiosis, production of lytic enzymes, induction of systemic resistance in host plant, and competition for nutrients or space. However, the precise mechanism of biocontrol of the blast fungus by antagonistic microorganisms and/or their bioactive secondary metabolites is not well understood. Commercial formulations of biocontrol agents and bioactive natural products could be cost-effective and sustainable but their availability at this time is extremely limited. This review updates our knowledge on the infection pathway of the wheat blast fungus, catalogs naturally occurring biocontrol agents that may be effective against blast diseases, and discusses their role in sustainable management of the disease.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

Accumulation of Catechin and Proanthocyanidins in Black Poplar Stems After Infection by Plectosphaerella populi: Hormonal Regulation, Biosynthesis and Antifungal Activity.

Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.

Sclerotinia sclerotiorum Circumvents Flavonoid Defenses by Catabolizing Flavonol Glycosides and Aglycones.

Flavonols are widely distributed plant metabolites that inhibit microbial growth. Yet many pathogens cause disease in flavonol-containing plant tissues. We investigated how Sclerotinia sclerotiorum, a necrotrophic fungal pathogen that causes disease in a range of economically important crop species, is able to successfully infect flavonol-rich tissues of Arabidopsis (Arabidopsis thaliana). Infection of rosette stage Arabidopsis with a virulent S. sclerotiorum strain led to the selective hydrolysis of flavonol glycosidic linkages and the inducible degradation of flavonol aglycones to phloroglucinol carboxylic and phenolic acids. By chemical analysis of fungal biotransformation products and a search of the S. sclerotiorum genome sequence, we identified a quercetin dioxygenase gene (QDO) and characterized the encoded protein, which catalyzed cleavage of the flavonol carbon skeleton. QDO deletion lines degraded flavonols with much lower efficiency and were less pathogenic on Arabidopsis leaves than wild-type S. sclerotiorum, indicating the importance of flavonol degradation in fungal virulence. In the absence of QDO, flavonols exhibited toxicity toward S. sclerotiorum, demonstrating the potential roles of these phenolic compounds in protecting plants against pathogens.

Strigolactones enhance root-knot nematode (Meloidogyne graminicola) infection in rice by antagonizing the jasmonate pathway.

Strigolactones (SLs) are carotenoid-derived plant hormones that also act in the rhizosphere to stimulate germination of root-parasitic plants and enhance plant symbiosis with beneficial microbes. Here, the role of SLs was investigated in the interaction of rice (Oryza sativa) roots with the root-knot nematode Meloidogyne graminicola. Genetic approaches and chemical sprays were used to manipulate SL signaling in rice before infection with M. graminicola. Then, nematode performance was evaluated and plant defense hormones were quantified. Meloidogyne graminicola infection induced SL biosynthesis and signaling and suppressed jasmonic acid (JA)-based defense in rice roots, suggesting a potential role of SLs during nematode infection. Whereas the application of a low dose of the SL analogue GR24 increased nematode infection and decreased jasmonate accumulation, the SL biosynthesis and signaling d mutants were less susceptible to M. graminicola, and constitutively accumulated JA and JA-isoleucine compared with wild-type plants. Spraying with 0.1 µM GR24 restored nematode susceptibility in SL-biosynthesis mutants but not in the signaling mutant. Furthermore, foliar application of the SL biosynthesis inhibitor TIS108 impeded nematode infection and increased jasmonate levels in rice roots. In conclusion, SL signaling in rice suppresses jasmonate accumulation and promotes root-knot nematode infection.

Flavanone-3-Hydroxylase Plays an Important Role in the Biosynthesis of Spruce Phenolic Defenses Against Bark Beetles and Their Fungal Associates.

Conifer forests worldwide are becoming increasingly vulnerable to attacks by bark beetles and their fungal associates due to the effects of global warming. Attack by the bark beetle Ips typographus and the blue-stain fungus it vectors (Endoconidiophora polonica) on Norway spruce (Picea abies) is well known to induce increased production of terpene oleoresin and polyphenolic compounds. However, it is not clear whether specific compounds are important in resisting attack. In this study, we observed a significant increase in dihydroflavonol and flavan-3-ol content after inoculating Norway spruce with the bark beetle vectored fungus. A bioassay revealed that the dihydroflavonol taxifolin and the flavan-3-ol catechin negatively affected both I. typographus and E. polonica. The biosynthesis of flavan-3-ols is well studied in Norway spruce, but little is known about dihydroflavonol formation in this species. A flavanone-3-hydroxylase (F3H) was identified that catalyzed the conversion of eriodictyol to taxifolin and was highly expressed after E. polonica infection. Down-regulating F3H gene expression by RNA interference in transgenic Norway spruce resulted in significantly lower levels of both dihydroflavonols and flavan-3-ols. Therefore F3H plays a key role in the biosynthesis of defense compounds in Norway spruce that act against the bark beetle-fungus complex. This enzyme forms a defensive product, taxifolin, which is also a metabolic precursor of another defensive product, catechin, which in turn synergizes the toxicity of taxifolin to the bark beetle associated fungus.

Salicylic acid activates poplar defense against the biotrophic rust fungus Melampsora larici-populina via increased biosynthesis of catechin and proanthocyanidins.

Poplar trees synthesize flavan-3-ols (catechin and proanthocyanidins) as a defense against foliar rust fungi, but the regulation of this defense response is poorly understood. Here, we investigated the role of hormones in regulating flavan-3-ol accumulation in poplar during rust infection. We profiled levels of defense hormones, signaling genes, and flavan-3-ol metabolites in black poplar leaves at different stages of rust infection. Hormone levels were manipulated by external sprays, genetic engineering, and drought to reveal their role in rust fungal defenses. Levels of salicylic acid (SA), jasmonic acid, and abscisic acid increased in rust-infected leaves and activated downstream signaling, with SA levels correlating closely with those of flavan-3-ols. Pretreatment with the SA analog benzothiadiazole increased flavan-3-ol accumulation by activating the MYB-bHLH-WD40 complex and reduced rust proliferation. Furthermore, transgenic poplar lines overproducing SA exhibited higher amounts of flavan-3-ols constitutively via the same transcriptional activation mechanism. These findings suggest a strong association among SA, flavan-3-ol biosynthesis, and rust resistance in poplars. Abscisic acid also promoted poplar defense against rust infection, but likely through stomatal immunity independent of flavan-3-ols. Jasmonic acid did not confer any apparent defense responses to the fungal pathogen. We conclude that SA activates flavan-3-ol biosynthesis in poplar against rust infection.

Flavan-3-ols Are an Effective Chemical Defense against Rust Infection.

Phenolic secondary metabolites are often thought to protect plants against attack by microbes, but their role in defense against pathogen infection in woody plants has not been investigated comprehensively. We studied the biosynthesis, occurrence, and antifungal activity of flavan-3-ols in black poplar (Populus nigra), which include both monomers, such as catechin, and oligomers, known as proanthocyanidins (PAs). We identified and biochemically characterized three leucoanthocyanidin reductases and two anthocyanidin reductases from P. nigra involved in catalyzing the last steps of flavan-3-ol biosynthesis, leading to the formation of catechin [2,3-trans-(+)-flavan-3-ol] and epicatechin [2,3-cis-(-)-flavan-3-ol], respectively. Poplar trees that were inoculated with the biotrophic rust fungus (Melampsora larici-populina) accumulated higher amounts of catechin and PAs than uninfected trees. The de novo-synthesized catechin and PAs in the rust-infected poplar leaves accumulated significantly at the site of fungal infection in the lower epidermis. In planta concentrations of these compounds strongly inhibited rust spore germination and reduced hyphal growth. Poplar genotypes with constitutively higher levels of catechin and PAs as well as hybrid aspen (Populus tremula × Populus alba) overexpressing the MYB134 transcription factor were more resistant to rust infection. Silencing PnMYB134, on the other hand, decreased flavan-3-ol biosynthesis and increased susceptibility to rust infection. Taken together, our data indicate that catechin and PAs are effective antifungal defenses in poplar against foliar rust infection.

O-Acyl Sugars Protect a Wild Tobacco from Both Native Fungal Pathogens and a Specialist Herbivore.

O-Acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. We characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore Manduca sexta At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A 3-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination, respectively. Finally, silencing the expression of a putative branched-chain α-ketoacid dehydrogenase E1 ß-subunit-encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20% to 30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack by both native pathogenic fungi and a specialist herbivore and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

The role of thionins in rice defence against root pathogens.

Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in-depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root-knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differentially regulated by defence-related hormones, whereas all analysed genes were consistently down-regulated in M. graminicola-induced galls, at least until 7 days post-inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 revealed decreased susceptibility to M. graminicola infection and P. graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in defence against two of the most damaging root pathogens attacking rice.