Phytohormone Priming of Tomato Plants Evoke Differential Behavior in Rhizoctonia solani During Infection, With Salicylate Priming Imparting Greater Tolerance Than Jasmonate

In the field of phytohormone defense, the general perception is that salicylate (SA)-mediated defense is induced against biotrophic pathogens while jasmonate (JA)-mediated defense functions against necrotrophic pathogens. Our goals were to observe the behavior of the necrotrophic pathogen Rhizoctonia solani in the vicinity, on the surface, and within the host tissue after priming the host with SA or JA, and to see if priming with these phytohormones would affect the host defense differently upon infection. It was observed for the first time, that R. solani could not only distinguish between JA versus SA-primed tomato plants from a distance, but surprisingly avoided SA-primed plants more than JA-primed plants. To corroborate these findings, early infection events were monitored and compared through microscopy, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy using transformed R. solani expressing green fluorescence protein gene (gfp). Different histochemical and physiological parameters were compared between the unprimed control, JA-primed, and SA-primed plants after infection. The expression of a total of fifteen genes, including the appressoria-related gene of the pathogen and twelve marker genes functioning in the SA and JA signaling pathways, were monitored over a time course during early infection stages. R. solani being traditionally designated as a necrotroph, the major unexpected observations were that Salicylate priming offered better tolerance than Jasmonate priming and that it was mediated through the activation of SA-mediated defense during the initial phase of infection, followed by JA-mediated defense in the later phase. Hence, the present scenario of biphasic SA-JA defense cascades during R. solani infection, with SA priming imparting maximum tolerance, indicate a possible hemibiotrophic pathosystem that needs to be investigated further.

Relationship of Resistance-Related Enzyme activity and Salicylic Acid content in Phalaenopsis Species with different levels of resistance to DIckeya Dadantii

ABSTRACT Orchids (Phalaenopsis) are ornamental plants that are cultivated commercially and in great demand in the market. Soft-rot disease (SRD) caused by the necrotrophic pathogen Dickeya dadantii is a cause of considerable economic loss to cultivators of many orchid species. Our previous experiment identified a limited number of species that were resistant to D. dadantii. This study aimed to validate the resistance level of four Phalaenopsis species in a detached leaf inoculation protocol to identify the resistance mechanism(s) involved. Soft-rot symptom diameter was measured from 6 to 18 hours post-inoculation (HPI) with D. dadantii. Disease assessment confirmed that P. amboinensis is a resistant species, P. pantherina is a susceptible species, and P. amabilis and P. schilleriana are very susceptible species. There was no difference in the lignin content between the resistant and very susceptible species. Detailed observation of resistant and very susceptible species, P. amboinensis vs. P. amabilis, revealed higher phenylalanine ammonia-lyase (PAL) and peroxidase (POD) in P. amabilis than in P. amboinensis. In contrast, there was higher salicylic acid (SA) content in P. amboinensis than in P. amabilis. These results suggest that POD and PAL activities may not be effective in defense against soft-rot disease, while SA plays an important role in the resistance of P. amboinensis to D. dadantii. Low PAL activity in P. amboinensis implies that the SA contents from the isochorismate pathway may be involved in the mechanism of P. amboinensis resistance to D. dadantii. Therefore, endogenous SA content may be a good indicator for screening resistant species in Phalaenopsis.

The Effect of Monochromatic LED Light Wavelengths and Photoperiods on Botrytis cinerea

Botrytis cinerea is a ubiquitous necrotrophic pathogen causing grey mould in economically important crops. Light effect in horticulture is undeniable and fungi also react to light. Selected specific light-emitting diodes (LEDs) and photoperiods can be used for fungal pathogen inhibition. This study aimed to evaluate how LED light wavelengths and photoperiods affect the growth parameters of B. cinerea. The morphological (mycelium appearance, sclerotia distribution) and phenotypic (conidia presence and size, mycelium growth rate, recovery) characteristics of the fungal pathogen B. cinerea were evaluated under royal blue 455 nm, blue 470 nm, cyan 505 nm, yellow 590 nm, and red 627 nm LED lights at various photoperiods (4, 8, 12, 16, 20, 24 h). The results revealed that the light conditions and photoperiods influenced the B. cinerea morphological and phenotypic characteristics. Overall, the highest B. cinerea inhibition was under yellow (590 nm) LED light at 4 and 8 h photoperiods. Conidia did not form under blue 455 nm at 8, 16, 20, and 24 h photoperiods. Therefore, it can be assumed that the phenotypic and morphological features of B. cinerea depend on the specific photoperiod and LED light wavelength. The results allowed an exploration of original research approaches, raised new scientific questions for further investigation, and suggested new green plant protection solutions.

The THO/TREX Complex Active in Alternative Splicing Mediates Plant Responses to Salicylic Acid and Jasmonic Acid

Salicylic acid (SA) and jasmonic acid (JA) are essential plant immune hormones, which could induce plant resistance to multiple pathogens. However, whether common components are employed by both SA and JA to induce defense is largely unknown. In this study, we found that the enhanced disease susceptibility 8 (EDS8) mutant was compromised in plant defenses to hemibiotrophic pathogen Pseudomonas syringae pv. maculicola ES4326 and necrotrophic pathogen Botrytis cinerea, and was deficient in plant responses to both SA and JA. The EDS8 was identified to be THO1, which encodes a subunit of the THO/TREX complex, by using mapping-by-sequencing. To check whether the EDS8 itself or the THO/TREX complex mediates SA and JA signaling, the mutant of another subunit of the THO/TREX complex, THO3, was tested. THO3 mutation reduced both SA and JA induced defenses, indicating that the THO/TREX complex is critical for plant responses to these two hormones. We further proved that the THO/TREX interacting protein SERRATE, a factor regulating alternative splicing (AS), was involved in plant responses to SA and JA. Thus, the AS events in the eds8 mutant after SA or JA treatment were determined, and we found that the SA and JA induced different alternative splicing events were majorly modulated by EDS8. In summary, our study proves that the THO/TREX complex active in AS is involved in both SA and JA induced plant defenses.

Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen – Alternaria brassicae

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

Transcriptome Analysis of Potato Infected with the Necrotrophic Pathogen Alternaria solani

Potato early blight is caused by the necrotrophic fungus Alternaria solani and can result in yield losses of up to 50% if left uncontrolled. At present, the disease is controlled by chemical fungicides, yet rapid development of fungicide resistance renders current control strategies unsustainable. On top of that, a lack of understanding of potato defences and the quantitative nature of resistance mechanisms against early blight hinders the development of more sustainable control methods. Necrotrophic pathogens, compared to biotrophs, pose an extra challenge to the plant, since common defence strategies to biotic stresses such as the hypersensitive response and programmed cell death are often beneficial for necrotrophs. With the aim of unravelling plant responses to both the early infection stages (i.e., before necrosis), such as appressorium formation and penetration, as well as to later responses to the onset of necrosis, we present here a transcriptome analysis of potato interactions with A. solani from 1 h after inoculation when the conidia have just commenced germination, to 48 h post inoculation when multiple cell necrosis has begun. Potato transcripts with putative functions related to biotic stress tolerance and defence against pathogens were upregulated, including a putative Nudix hydrolase that may play a role in defence against oxidative stress. A. solani transcripts encoding putative pathogenicity factors, such as cell wall degrading enzymes and metabolic processes that may be important for infection. We therefore identified the differential expression of several potato and A. solani transcripts that present a group of valuable candidates for further studies into their roles in immunity or disease development.

ε-poly-L-lysine Affects the Vegetative Growth, Pathogenicity and Expression Regulation of Necrotrophic Pathogen Sclerotinia sclerotiorum and Botrytis cinerea

Microbial secondary metabolites produced by Streptomyces are applied to control plant diseases. The metabolite, ε-poly-L-lysine (ε-PL), is a non-toxic food preservative, but the potential application of this compound as a microbial fungicide in agriculture is rarely reported. In this study, the effect and mode of action of ε-PL on two necrotrophic pathogenic fungi, Sclerotinia sclerotiorum and Botrytis cinerea, were investigated. The results showed that ε-PL effectively inhibited the mycelial growth of S. sclerotiorum and B. cinerea with EC50 values of 283 μg/mL and 281 μg/mL, respectively. In addition, ε-PL at the dose of 150 and 300 μg/mL reduced S. sclerotiorum sclerotia formation. The results of the RNA-seq and RT-qPCR validation indicated that ε-PL significantly regulated the gene expression of critical differential expressed genes (DEGs) involved in fungal growth, metabolism, pathogenicity, and induced an increase in the expression of the fungal stress responses and the detoxification genes. These results provided new insights for understanding the modes of action of ε-PL on S. sclerotiorum and B. cinerea and improved the sustainable management of these plant diseases.

MPK3/MPK6-mediated ERF72 Phosphorylation Positively Regulates Resistance to Botrytis cinerea Through Directly and Indirectly Activating the Transcription of Camalexin-biosynthesis Enzymes

Ethylene response factor (ERF) Group VII members generally function in regulating plant growth and development, abiotic stress response, and plant immunity in Arabidopsis. However, the detail regulatory mechanism by which Group VII ERFs mediate plant immune responses remains elusive. Here, we characterised ERF72, a member of the Group VII ERFs, as a positive regulator mediating resistance to the necrotrophic pathogen Botrytis cinerea. Compared with wild type (WT), erf72 mutant showed the lower camalexin contents and more susceptible to B. cinerea, while complementation of ERF72 in erf72 rescued susceptibility phenotypes. Moreover, overexpression of ERF72 in WT promoted camalexin biosynthesis and resistance to B. cinerea. Then, we identified camalexin biosynthesis genes PAD3 and CYP71A13, and transcription factor WRKY33 as target genes of ERF72. Furthermore, MPK3 and MPK6 phosphorylate ERF72 at Ser151 to improve its transactivation activity, camalexin contents and resistance to B. cinerea. These findings highlight the role of ERF72 in coordinating the camalexin biosynthesis via directly regulating the expression of camalexin biosynthetic genes and indirectly by targeting WRKK33 in plant immunity.

The Ca2+ sensor proteins CML37 and CML42 antagonistically regulate plant stress responses by altering phytohormone signals

Key message Calmodulin-like-proteins (CML) belong to a family of calcium-sensing proteins that are unique for plants and involved in many different developmental and stress-related reactions. In defense against herbivory, some pathogens and drought, CML37 acts as a positive and CML42 as a negative regulator, respectively. We provide evidence that both CMLs act antagonistically in the regulation of induced defense responses. A double knock-out line, cml37 x cml42, thus shows wild-type phenotypes upon all kind of stresses we used. Abstract A transient increase in the cytosolic calcium concentration is one of the first reactions that can be measured in plant cells upon abiotic as well as biotic stress treatments. These calcium signals are sensed by calcium binding proteins such as calmodulin-like proteins (CMLs), which transduce the sensed information into appropriate stress responses by interacting with downstream target proteins. In previous studies, CML37 has been shown to positively regulate the plants’ defense against both the insect herbivore Spodoptera littoralis and the response to drought stress. In contrast, CML42 is known to negatively regulate those two stress responses. Here, we provide evidence that these two CMLs act antagonistically in the regulation of induced responses directed against drought and herbivory stress as well as in the defense against the necrotrophic pathogen Alternaria brassicicola. Both CMLs shape the plant reactions by altering the phytohormone signaling. Consequently, the phytohormone-regulated production of defensive compounds like glucosinolates is also antagonistically mediated by both CMLs. The finding that CML37 and CML42 have antagonistic roles in diverse stress-related responses suggests that these calcium sensor proteins represent important tools for the plant to balance and fine-tune the signaling and downstream reactions upon environmental stress.