SsPEP1, an Effector with Essential Cellular Functions in Sugarcane Smut Fungus

Biotrophic fungi have to infect their host to obtain nutrients and must establish an interaction with the host to complete their life cycle. In this process, effectors play important roles in manipulating the host’s immune system to avoid being attacked. Sporisorium scitamineum is the causative agent of sugarcane smut, the most important disease in sugarcane-producing regions worldwide. In this work, we functionally characterized the conserved effector PEP1 in S. scitamineum. The mating process and the expression of genes in the MAPK signaling pathway and the a and b loci were adversely affected in Sspep1-null mutants. The requirement for SsPEP1 in pathogenicity and symptom development was allele dosage-dependent, i.e., deleting one Sspep1 allele in the mating pair turned a normal black whip with abundant teliospores into a white whip with few teliospores; however, deleting both alleles almost abolished infectivity and whip development. ΔSspep1 mutants produced significantly less mycelium mass within infected plants. Additionally, SsPEP1 was identified as a potent inhibitor of sugarcane POD-1a peroxidase activity, implying that SsPEP1 may function to relieve reactive oxygen species-related stress within the host plant. Taken together, our work demonstrated that SsPEP1 is a multifaceted effector essential for S. scitamineum growth, development, and pathogenicity.

Maize AFP1 confers antifungal activity by inhibiting chitin deacetylases from a broad range of fungi

Adapted plant pathogenic fungi deacetylate chitin to chitosan to avoid host perception and disarm the chitin-triggered plant immunity. Whether plants have evolved factors to counteract this fungal evasion mechanism in the plant-pathogen interface remains obscure. Here, we decipher the underlying mechanism of maize cysteine-rich receptor-like secreted proteins (CRRSPs)- AFP1, which exhibits mannose-binding dependent antifungal activity. AFP1 initials the action by binding to specific sites on the surface of yeast-like cells, filaments, and germinated spores of the biotrophic fungi Ustilago maydis. This could result in fungal cell growth and cell budding inhibition, delaying spore germination and subsequently reducing fungal viability in a mannose-binding dependence manner. The antifungal activity of AFP1 is conferred by its interaction with the PMT-dependent mannosylated chitin deacetylases (CDAs) and interfering with the conversion of chitin. Our finding that AFP1 targets CDAs from pathogenic fungi and nonpathogenic budding yeast suggests a potential application of the CRRSP in combating fungal diseases and reducing threats posed by the fungal kingdom.

The Barley HvSTP13GR mutant triggers resistance against biotrophic fungi

High-yielding and stress resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, that possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analyzed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased upon bacterial and fungal PAMP application, suggesting a PAMP-triggered signaling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is likely targeted by transcription factors downstream of PAMP-triggered immunity pathways. We confirmed that the non-functional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus, in barley. In addition, we established targeted CRISPR/Cas9 cytosine base editing in barley protoplasts to generate alternative HvSTP13 mutants and characterized the sugar transport activity and subcellular localization of the proteins. These mutants represent promising variants for future resistance analysis. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host pathogen interaction, is a promising approach to achieve broad and durable resistance in plants.

Influence of Interactions between Nitrogen, Phosphorus Supply and Epichloёbromicola on Growth of Wild Barley (Hordeum brevisubulatum)

Epichloë endophytes are biotrophic fungi that establish mutualistic symbiotic relationship with grasses and affect performance of the host under different environments. Wild barley (Hordeum brevisubulatum) is an important forage grass and often infected by Epichloë bromicola, thus showing tolerances to stresses. Since the plant growth correlates with both microbial infection and nutrient stoichiometry, this study was performed to investigate whether the function of Epichloë bromicola endophyte to improve host growth depend upon the nitrogen (N), phosphorus (P) fertilization. Epichloë-infected (E+) and Epichloë-free (E−) wild barley plants were subjected to nine types of mixed N (0.2 mM, 3 mM, 15 mM) and P (0.01 mM, 0.1 mM, 1.5 mM) levels treatments for 90 d to collect plant samples and determine multiple related indexes. We found that E. bromicola and N, P additions positively affected seed germination. Further, E. bromicola significantly enhanced chlorophyll content and root metabolic activity under N-deficiency, and meanwhile, might alter allocation of photosynthate under different conditions. The contents of N, P and stoichiometry of C:N:P of E+ plants were significantly higher than that of E− under nutrient deficiency, but contrary results were observed under adequate nutrients. Therefore, we propose that the growth-promoting ability of E. bromicola is closely correlated with N and P additional levels. Under low N, P additions, positive roles of endophyte are significant as opposed to negative roles under high N, P additions.

EffectorP 3.0: prediction of apoplastic and cytoplasmic effectors in fungi and oomycetes

Many fungi and oomycete species are devasting plant pathogens. These eukaryotic filamentous pathogens secrete effector proteins to facilitate plant infection. Fungi and oomycete pathogens have diverse infection strategies and their effectors generally do not share sequence homology. However, they occupy similar host environments, either the plant apoplast or plant cytoplasm, and may therefore share some unifying properties based on the requirements of these host compartments. Here we exploit these biological signals and present the first classifier (EffectorP 3.0) that uses two machine learning models: one trained on apoplastic effectors and one trained on cytoplasmic effectors. EffectorP 3.0 accurately predicts known apoplastic and cytoplasmic effectors in fungal and oomycete secretomes with low estimated false positive rates of 3% and 8%, respectively. Cytoplasmic effectors have a higher proportion of positively charged amino acids, whereas apoplastic effectors are enriched for cysteine residues. The combination of fungal and oomycete effectors in training leads to a higher number of predicted cytoplasmic effectors in biotrophic fungi. EffectorP 3.0 expands predicted effector repertoires beyond small, cysteine-rich secreted proteins in fungi and RxLR-motif containing secreted proteins in oomycetes. We show that signal peptide prediction is essential for accurate effector prediction, as EffectorP 3.0 recognizes a cytoplasmic signal also in intracellular, non-secreted proteins. EffectorP 3.0 is available at http://effectorp.csiro.au.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate

Xylem sap is the major transport route for nutrients from roots to shoots. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, while nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased whereas salicylic acid and jasmonoyl-isoleucine decreased with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Homeopathy in the Rust Severity and Growth of Malva sylvestris L.

The production of medicinal plants which have an association with biotrophic fungi requires non-residual and favorable methods to the host with tolerance to the presence of phytopathogens. The objective of this work was to evaluate the effect of homeopathic preparations on the rust severity and the growth of Malva sylvestris plants. M. sylvestris seedlings were prepared in 600 ml containers with commercial substrate. The seedlings were arranged in pots at 26 days of age and outlined in two experiments. The treatments consisted of Amonnium carbonicum (Am. carb.), Atropa belladonna (Bell.), Calcarea carbonica (Calc. carb.), Silicea terra (Sil.) and Sulfur (Sulf.), all at 30CH (centesimal Hahnemannian dilution order). The last two dynamizations (29 and 30CH) were prepared in distilled water for all treatments. Control plants were treated with water. Natural inoculation of the plants with Puccinia malvacearum occurred in the first experiment, and the applications of homeopathic preparations were carried out every seven days for five weeks. Four evaluations of rust severity, diameter, height and number of leaves were conducted. Next, M. sylvestris seedlings were transplanted into pots with 5 liters of substrate in the second experiment and the growth curve of the plant was observed in relation to the diameter and height variables. Am. Carb. reduced 18.29% of the rust severity in relation to the control plants. Sil. 30CH contributed to an increase in stem diameter. There was no interference in the plants’ height by homeopathic preparations. The application of homeopathies in M. sylvestris can contribute to their production, reducing the rust intensity considered in the crop cycle and can assist in the plant growth without leaving residues which can harm pollinators and hyperparasites.

A Hyperspectral Library of Foliar Diseases of Wheat

This work established a hyperspectral library of important foliar diseases of wheat in time series to detect spectral changes from infection to symptom appearance induced by different pathogens. The data was generated under controlled conditions at the leaf-scale. The transition from healthy to diseased leaf tissue was assessed, spectral shifts were identified and used in combination with histological investigations to define developmental stages in pathogenesis for each disease. The spectral signatures of each plant disease that are indicative of a certain developmental stage during pathogenesis - defined as turning points - were combined into a spectral library. Different machine learning analysis methods were applied and compared to test the potential of this library for the detection and quantification of foliar diseases in hyperspectral images. All evaluated classifiers provided a high accuracy for the detection and identification for both the biotrophic fungi and the necrotrophic fungi of up to 99%. The potential of applying spectral analysis methods, in combination with a spectral library for the detection and identification of plant diseases is demonstrated. Further evaluation and development of these algorithms should contribute to a robust detection and identification system for plant diseases at different developmental stages and the promotion and development of site-specific management techniques of plant diseases under field conditions.

WY195, a New Inducible Promoter From the Rubber Powdery Mildew Pathogen, Can Be Used as an Excellent Tool for Genetic Engineering

Until now, there are few studies and reports on the use of endogenous promoters of obligate biotrophic fungi. The WY195 promoter in the genome of Oidium heveae, the rubber powdery mildew pathogen, was predicted using PromoterScan and its promoter function was verified by the transient expression of the β-glucuronidase (GUS) gene. WY195 drove high levels of GUS expression in dicotyledons and monocotyledons. qRT-PCR indicated that GUS expression regulated by the WY195 promoter was 17.54-fold greater than that obtained using the CaMV 35S promoter in dicotyledons (Nicotiana tabacum), and 5.09-fold greater than that obtained using the ACT1 promoter in monocotyledons (Oryza sativa). Furthermore, WY195-regulated GUS gene expression was induced under high-temperature and drought conditions. Soluble proteins extracted from WY195-hpaXm transgenic tobacco was bioactive. Defensive micro-HR induced by the transgene expression of hpaXm was observed on transgenic tobacco leaves. Disease resistance bioassays showed that WY195-hpaXm transgenic tobacco enhanced the resistance to tobacco mosaic virus (TMV). WY195 has great potential for development as a new tool for genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms and the pathogenic mechanisms of O. heveae.