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Pearl millet [Pennisetum glaucum (L.) R. Br.] is the essential food crop for over ninety million people living in drier parts of India and South Africa. Pearl millet crop production is harshly hindered by numerous biotic stresses. Sclerospora graminicola causes downy mildew disease in pearl millet. Effectors are the proteins secreted by several fungi and bacteria that manipulate the host cell structure and function. This current study aims to identify genes encoding effector proteins from the S. graminicola genome and validate them through molecular techniques. In silico analyses were employed for candidate effector prediction. A total of 845 secretory transmembrane proteins were predicted, out of which 35 proteins carrying LxLFLAK (Leucine–any amino acid–Phenylalanine–Leucine–Alanine–Lysine) motif were crinkler, 52 RxLR (Arginine, any amino acid, Leucine, Arginine), and 17 RxLR-dEER putative effector proteins. Gene validation analysis of 17 RxLR-dEER effector protein-producing genes was carried out, of which 5genes were amplified on the gel. These novel gene sequences were submitted to NCBI. This study is the first report on the identification and characterization of effector genes in Sclerospora graminicola. This dataset will aid in the integration of effector classes that act independently, paving the way to investigate how pearl millet responds to effector protein interactions. These results will assist in identifying functional effector proteins involving the omic approach using newer bioinformatics tools to protect pearl millet plants against downy mildew stress. Considered together, the identified effector protein-encoding functional genes can be utilized in screening oomycetes downy mildew diseases in other crops across the globe.

A wide variety of DNA nanostructures have been assembled by folding long DNA single strands with short DNA staples. However, such structures typically need annealing at elevated temperatures in order to form. To accommodate the formation of such structures in living cells, Praetorius and Dietz developed an approach in which custom protein staples based on transcription activator–like effector proteins fold double-stranded DNA templates (see the Perspective by Douglas). The structures folded into user-defined geometric shapes on the scale of tens to hundreds of nanometers. These nanostructures could self-assemble at room temperature in physiological buffers. Science , this issue p. eaam5488 ; see also p. 1261 Folding DNA double strands using custom staple proteins can form megadalton-scale nanostructures with user-defined shapes. We describe an approach to bottom-up fabrication that allows integration of the functional diversity of proteins into designed three-dimensional structural frameworks. A set of custom staple proteins based on transcription activator–like effector proteins folds a double-stranded DNA template into a user-defined shape. Each staple protein is designed to recognize and closely link two distinct double-helical DNA sequences at separate positions on the template. We present design rules for constructing megadalton-scale DNA-protein hybrid shapes; introduce various structural motifs, such as custom curvature, corners, and vertices; and describe principles for creating multilayer DNA-protein objects with enhanced rigidity. We demonstrate self-assembly of our hybrid nanostructures in one-pot mixtures that include the genetic information for the designed proteins, the template DNA, RNA polymerase, ribosomes, and cofactors for transcription and translation.

The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively reenter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.

Dothistroma needle blight, Cyclaneusma needle blight and red needle cast are devastating foliar pine diseases caused by the fungi Dothistroma septosporum and Cyclaneusma minus and the oomycete Phytophthora pluvialis, respectively. These pathogens colonise the host apoplast, secreting effector proteins to promote infection and disease. If these effectors are recognised by corresponding host resistance proteins, they activate the plant immune system to stop pathogen growth. We aimed to identify and characterise effectors that are common to all three pathogens. Using D. septosporum as a starting point, three candidate effectors (CEs) were investigated: Ds69335 (a CAP protein) and Ds131885, both of which have sequence and structural similarity to CEs of C. minus and P. pluvialis, and Ds74283, which adopts a β-trefoil fold and has structural rather than sequence similarity to CEs from all three pathogens. Notably, of the CEs investigated, Ds74283 and Ds131885, as well as their homologues from C. minus and P. pluvialis, elicited chlorosis or cell death in Nicotiana species, with Ds131885 and its homologues also triggering cell death in Pinus radiata. In line with these observed responses being related to activation of the plant immune system, the chlorosis triggered by Ds131885 and its homologues was compromised in a Nicotiana benthamiana mutant lacking the extracellular immune system co-receptor, SOBIR1. Such cross-kingdom, plant immune system-activating effectors, whether similar in sequence or structure, might ultimately enable the selection or engineering of durable, broad-spectrum resistance against foliar pine pathogens

Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein X-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici ( Fol ). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol- tomato, and by extension plant–fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens.

Over the past decade, tremendous progress has been made in plant pathology, broadening our understanding of how pathogens colonize their hosts. To manipulate host cell physiology and subvert plant immune responses, pathogens secrete an array of effector proteins. A coevolutionary arms-race drives the pathogen to constantly reinvent its effector repertoire to undermine plant immunity. In turn, hosts develop novel immune receptors to maintain effector recognition and mount defences. Understanding how effectors promote disease and how they are perceived by the plant’s defence network persist as major subjects in the study of plant–pathogen interactions. Here, we focus on recent advances (over roughly the last two years) in understanding structure/function relationships in effectors from bacteria and filamentous plant pathogens. Structure/function studies of bacterial effectors frequently uncover diverse catalytic activities, while structure-informed similarity searches have enabled cataloguing of filamentous pathogen effectors. We also suggest how such advances have informed the study of plant–pathogen interactions.

This article is a Commentary on Wang et al., 216: 205–215.

Plant diseases caused by necrotrophic fungal pathogens result in large economic losses in field crop production worldwide. Effectors are important players of plant-pathogen interaction and deployed by pathogens to facilitate plant colonization and nutrient acquisition. Compared to biotrophic and hemibiotrophic fungal pathogens, effector biology is poorly understood for necrotrophic fungal pathogens. Recent bioinformatics advances have accelerated the prediction and discovery of effectors from necrotrophic fungi, and their functional context is currently being clarified. In this review we examine effectors utilized by necrotrophic fungi and hemibiotrophic fungi in the latter stages of disease development, including plant cell death manipulation. We define “effectors” as secreted proteins and other molecules that affect plant physiology in ways that contribute to disease establishment and progression. Studying and understanding the mechanisms of necrotrophic effectors is critical for identifying avenues of genetic intervention that could lead to improved resistance to these pathogens in plants.

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant–pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N‐terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine‐rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence‐derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high‐priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant–pathogen interactions. EffectorP is available at http://effectorp.csiro.au.

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB‐regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator‐like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA‐binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector‐binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5′ terminal guanine, contrasting with the TALE preference for a 5′ thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.