Plants and Microbes

Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.

Plant and animal nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins often function in pairs to mediate innate immunity to pathogens. However, the degree to which NLR proteins form signaling networks beyond genetically linked pairs is poorly understood. In this study, we discovered that a large NLR immune signaling network with a complex genetic architecture confers immunity to oomycetes, bacteria, viruses, nematodes, and insects. The network emerged over 100 million years ago from a linked NLR pair that diversified into up to one half of the NLR of asterid plants. We propose that this NLR network increases robustness of immune signaling to counteract rapidly evolving plant pathogens.

The CRISPR/Cas9 system has recently been used to confer molecular immunity against several eukaryotic viruses, including plant DNA geminiviruses. Here, we provide a detailed analysis of the efficiencies of targeting different coding and non-coding sequences in the genomes of multiple geminiviruses. Moreover, we analyze the ability of geminiviruses to evade the CRISPR/Cas9 machinery. Our results demonstrate that the CRISPR/Cas9 machinery can efficiently target coding and non-coding sequences and interfere with various geminiviruses. Furthermore, targeting the coding sequences of different geminiviruses resulted in the generation of viral variants capable of replication and systemic movement. By contrast, targeting the noncoding intergenic region sequences of geminiviruses resulted in interference, but with inefficient recovery of mutated viral variants, which thus limited the generation of variants capable of replication and movement. Taken together, our results indicate that targeting noncoding, intergenic sequences provides viral interference activity and significantly limits the generation of viral variants capable of replication and systemic infection, which is essential for developing durable resistance strategies for long-term virus control.

All filamentous microbes produce and release a wide range of glycans, which are essential determinants of microbe–microbe and microbe–host interactions. Major cell wall constituents, such as chitin and β-glucans, are elicitors of host immune responses. The widespread capacity for glycan perception in plants has driven the evolution of various strategies that help filamentous microbes to evade detection. Common strategies include structural and chemical modifications of cell wall components as well as the secretion of effector proteins that suppress chitin- and β-glucan-triggered immune responses. Thus, the necessity to avoid glycan-triggered immunity represents a driving force in the convergent evolution of filamentous microbes towards its suppression.

From 2006 through 2015, a research project on Durable Resistance in potato against Phytophthora (DuRPh) was carried out at Wageningen University and Research Centre. Its objective was to develop a proof of principle for durable resistance against late blight by cisgenesis. This public-funded project aimed at stimulating research on genetic modification and public debate on innovative genetic techniques. It was decided to clone and transfer late blight resistance (R) genes of crossable wild potato species (cisgenes) by Agrobacterium tumefaciens-mediated transformation without non-potato genes. A stack of multiple R genes were planned to be inserted into established varieties, thereby creating a dynamic variety in which the composition of the stacks may vary over space and time. Cisgenic plants were selected based on the expression of all inserted R genes and trueness-to-type. Within the project, 13 Rgenes from wild potato species were genetically mapped and three of them were cloned. Four varieties were transformed with one to three R genes. This was initially done using kanamycin resistance provided by a selectable marker gene of synthetic origin in order to quickly test the performance and stability of the introduced R genes and stacked R gene combinations. Once the functioning thereof was confirmed, marker-free transformations were conducted; thus, true cisgenic events were selected. The results about the different R genes, their chromosomal location, their specificity, the background dependence, the maximum size of a stack, its regeneration time and associated somaclonal variation frequency and its stability were studied. After selection and characterisation in the laboratory, the best cisgenic events were assessed in field trials for late blight resistance. This showed that inserted R genes were capable of turning a susceptible variety into a resistant one. Maximising longevity of the resistance was assured through resistance management research. It was shown that stacking of multiple R genes and monitoring how to deploy these stacks spatially and temporally could reduce fungicide use by over 80%. Communications through media and field demonstrations were manifold to allow public and policymakers to decide if cisgenesis is an acceptable tool to make potato farming more sustainable. Future deployment of the DuRPh strategy will depend largely on its status as a genetically modified crop or its exemption thereof. Worldwide near eradication of late blight would increase global annual potato production by close to 80 million tons, thereby contributing considerably to the needed additional global future food supply.

• The Phytophthora haustorium is a major site of secretion during infection.

• The host endocytic cycle contributes to biogenesis of the Extra-Haustorial Membrane.
• RXLR effectors manipulate host processes at diverse subcellular locations.

• They directly manipulate the activity or location of immune proteins.

• They also promote the activity of endogenous negative regulators of immunity.

Late blight, caused by the oomycete Phytophthora infestans, is a major global disease of potato and tomato. Cell biology is teaching us much about the developmental stages associated with infection, especially the haustorium, which is a site of intimate interaction and molecular exchange between pathogen and host. Recent observations suggest a role for the plant endocytic cycle in specific recruitment of host proteins to the Extra-Haustorial Membrane, emphasising the unique nature of this membrane compartment. In addition, there has been a strong focus on the activities of RXLR effectors, which are delivered into plant cells to modulate and manipulate host processes. RXLR effectors interact directly with diverse plant proteins at a range of subcellular locations to promote disease.

Magnaporthe oryzae, the fungus causing rice blast disease, should contend with host innate immunity to develop invasive hyphae (IH) within living host cells. However, molecular strategies to establish the biotrophic interactions are largely unknown. Here, we report the biological function of a M. oryzae-specific gene, Required-for-Focal-BIC-Formation 1 (RBF1). RBF1expression was induced in appressoria and IH only when the fungus was inoculated to living plant tissues. Long-term successive imaging of live cell fluorescence revealed that the expression of RBF1 was upregulated each time the fungus crossed a host cell wall. Like other symplastic effector proteins of the rice blast fungus, Rbf1 accumulated in the biotrophic interfacial complex (BIC) and was translocated into the rice cytoplasm. RBF1-knockout mutants (Δrbf1) were severely deficient in their virulence to rice leaves, but were capable of proliferating in abscisic acid-treated or salicylic acid-deficient rice plants. In rice leaves, Δrbf1 inoculation caused necrosis and induced defense-related gene expression, which led to a higher level of diterpenoid phytoalexin accumulation than the wild-type fungus did. Δrbf1 showed unusual differentiation of IH and dispersal of the normally BIC-focused effectors around the short primary hypha and the first bulbous cell. In the Δrbf1-invaded cells, symplastic effectors were still translocated into rice cells but with a lower efficiency. These data indicate that RBF1 is a virulence gene essential for the focal BIC formation, which is critical for the rice blast fungus to suppress host immune responses.

• Development of a fluorescence-based mitotic reporter strain of M. oryzae that expresses GFP-NLS together with H1-tdTomato.

• M. oryzae is hypothesized to undergo semi-closed mitosis accompanied by closure of septal pores.
• A nucleus undergoes extreme constriction and elongation during migration through the narrow invasive hyphal peg.

To study nuclear dynamics of Magnaporthe oryzae, we developed a novel mitotic reporter strain with GFP-NLS (localized in nuclei during interphase but in the cytoplasm during mitosis) and H1-tdTomato (localized in nuclei throughout the cell cycle). Time-lapse confocal microscopy of the reporter strain during host cell invasion provided several new insights into nuclear division and migration in M. oryzae: (i) mitosis lasts about 5 min; (ii) mitosis is semi-closed; (iii) septal pores are closed during mitosis; and (iv) a nucleus exhibits extreme constriction (approximately from 2 μm to 0.5 μm), elongation (over 5 μm), and long migration (over 16 μm). Our observations raise new questions about mechanisms controlling the mitotic dynamics, and the answers to these questions may result in new means to prevent fungal proliferation without negatively affecting the host cell cycle.

• Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive.
• Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations.
• The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor.
• Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors.

ITHACA, NY–Researchers have sequenced the genome of the whitefly (Bemisia tabici), an invasive insect responsible for spreading plant viruses worldwide, causing billions of dollars in crop losses each year.

The genome study, led by Associate Professor Zhangjun Fei of the Boyce Thompson Institute (BTI), offers many clues to the insect's remarkable ability to resist pesticides, transmit more than 300 plant viruses, and to feed on at least 1,000 different plant species. Published today in the journal BMC Biology, the study will serve as a foundation for future work to combat this global pest.

"Whitefly is an economically important pest for agriculture crops. It causes direct damage and also is a major vector for viruses, like Tomato yellow leaf curl virus, Cassava mosaic virus and Cassava brown streak virus, so it creates huge crop losses and poses serious threats to food security, especially in Africa and other parts of the developing world," said Fei.

In collaboration with a group of international colleagues, BTI researchers created a high-quality draft genome sequence of the whitefly and identified genes that code for proteins. The genome sequence can be accessed at the whitefly genome database developed by the Fei lab.

An analysis showed that, compared to related species, the whitefly has expanded families of detoxification genes. It also has extra genes that code for proteins related to virus acquisition and transmission, as well as insecticide resistance.

In an impressive example of horizontal gene transfer, the whitefly has acquired 142 genes from bacteria or fungi, including some coding for enzymes that break down foreign chemicals. These genes likely allow the whitefly to feed on diverse types of plants and to rapidly evolve resistance to insecticides.

Because pesticides are ineffective at keeping whitefly populations in check, collaborators at USDA plan to use the genome sequence to develop a control strategy using RNA interference (RNAi). Once scientists pinpoint the genes necessary for virus transmission and survival in the whitefly genome, they can develop new varieties of crops that will produce RNA molecules that block the expression of those necessary genes, killing the whitefly or preventing it from spreading the virus .

Microbial pathogens strategically acquire metabolites from their hosts during infection. Here we show that the host can intervene to prevent such metabolite loss to pathogens. Phosphorylation-dependent regulation of sugar transporter 13 (STP13) is required for antibacterial defense in the plant Arabidopsis thaliana. STP13 physically associates with the flagellin receptor flagellin-sensitive 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1). BAK1 phosphorylates STP13 at threonine 485, which enhances its monosaccharide uptake activity to compete with bacteria for extracellular sugars. Limiting the availability of extracellular sugar deprives bacteria of an energy source and restricts virulence factor delivery. Our results reveal that control of sugar uptake, managed by regulation of a host sugar transporter, is a defense strategy deployed against microbial infection. Competition for sugar thus shapes host-pathogen interactions.

High humidity has a strong influence on the development of numerous diseases affecting the above-ground parts of plants (the phyllosphere) in crop fields and natural ecosystems, but the molecular basis of this humidity effect is not understood. Previous studies have emphasized immune suppression as a key step in bacterial pathogenesis. Here we show that humidity-dependent, pathogen-driven establishment of an aqueous intercellular space (apoplast) is another important step in bacterial infection of the phyllosphere. Bacterial effectors, such as Pseudomonas syringae HopM1, induce establishment of the aqueous apoplast and are sufficient to transform non-pathogenic P. syringae strains into virulent pathogens in immunodeficient Arabidopsis thaliana under high humidity. Arabidopsis quadruple mutants simultaneously defective in a host target (AtMIN7) of HopM1 and in pattern-triggered immunity could not only be used to reconstitute the basic features of bacterial infection, but also exhibited humidity-dependent dyshomeostasis of the endophytic commensal bacterial community in the phyllosphere. These results highlight a new conceptual framework for understanding diverse phyllosphere–bacterial interactions.

Pathogenic fungi and oomycetes can establish intimate associations with plants. These interactions underlie a molecular dialogue that leads to the successful colonization of host tissues. Major questions driving research in plant pathology these last decades are how pathogenic microorganisms circumvent preformed or induced defences and how pathogens manipulate host physiology to promote virulence. One key actor in this dialogue relies on a class of molecules secreted by pathogens termed effectors. Effectors perturb host processes by targeting a variety of host functions either in the apoplast or in the cytosol of host cells. This chapter focuses on fungal and oomycetal cytoplasmic effectors by reviewing methods to predict and to characterize effectors as well as their activities and role during infection. We provide current knowledge regarding their evolution and their putative role in the shaping of plant-associated microbial communities.

The objective of this symposium is to create a scientific event that is at the forefront of fundamental research in beneficial plant-microbe interactions.

The symposium will bring together about 150 participants in a rather informal atmosphere, facilitating exchanges. We also aim at proposing a highly attractive program at a moderate inscription fee to give the opportunity to researchers - in particular those at the early stage of their career – to participate to an exciting top-level scientific event. Young researchers will have the opportunity to present their work with a poster.

Efficient strategies for limiting the impact of pathogens on crops require a good understanding of the factors underlying the evolution of compatibility range for the pathogens and host plants, i.e., the set of host genotypes that a particular pathogen genotype can infect and the set of pathogen genotypes that can infect a particular host genotype. Until now, little is known about the evolutionary and ecological factors driving compatibility ranges in systems implicating crop plants. We studied the evolution of host and pathogen compatibility ranges for rice blast disease, which is caused by the ascomycete Magnaporthe oryzae. We challenged 61 rice varieties from three rice subspecies with 31 strains of M. oryzae collected worldwide from all major known genetic groups. We determined the compatibility range of each plant variety and pathogen genotype and the severity of each plant−pathogen interaction. Compatibility ranges differed between rice subspecies, with the most resistant subspecies selecting for pathogens with broader compatibility ranges and the least resistant subspecies selecting for pathogens with narrower compatibility ranges. These results are consistent with a nested distribution of R genes between rice subspecies.

• Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive.
• Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors.
• We demonstrate translocation of a number of effectors in the U. maydis–maize system and show data that suggest that the uptake mechanism could be rather nonspecific
• The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established.