Infect Arabidopsis Thaliana Research Articles

Genome-Wide Characterization of Light-Regulated Gene Expression in Botrytis cinerea Reveals Underlying Complex Photobiology.

Botrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana.

TYMV and TRV infect Arabidopsis thaliana by expressing weak suppressors of RNA silencing and inducing host RNASE THREE LIKE1.

Post-Transcriptional Gene Silencing (PTGS) is a defense mechanism that targets invading nucleic acids of endogenous (transposons) or exogenous (pathogens, transgenes) origins. During plant infection by viruses, virus-derived primary siRNAs target viral RNAs, resulting in both destruction of single-stranded viral RNAs (execution step) and production of secondary siRNAs (amplification step), which maximizes the plant defense. As a counter-defense, viruses express proteins referred to as Viral Suppressor of RNA silencing (VSR). Some viruses express VSRs that totally inhibit PTGS, whereas other viruses express VSRs that have limited effect. Here we show that infection with the Turnip yellow mosaic virus (TYMV) is enhanced in Arabidopsis ago1, ago2 and dcl4 mutants, which are impaired in the execution of PTGS, but not in dcl2, rdr1 and rdr6 mutants, which are impaired in the amplification of PTGS. Consistently, we show that the TYMV VSR P69 localizes in siRNA-bodies, which are the site of production of secondary siRNAs, and limits PTGS amplification. Moreover, TYMV induces the production of the host enzyme RNASE THREE-LIKE 1 (RTL1) to further reduce siRNA accumulation. Infection with the Tobacco rattle virus (TRV), which also encodes a VSR limiting PTGS amplification, induces RTL1 as well to reduce siRNA accumulation and promote infection. Together, these results suggest that RTL1 could be considered as a host susceptibility gene that is induced by viruses as a strategy to further limit the plant PTGS defense when VSRs are insufficient.

Opposing effects of MYZUS PERSICAE-INDUCED LIPASE 1 and jasmonic acid influence the outcome of Arabidopsis thaliana-Fusarium graminearum interaction.

Fusarium graminearum (Fg) is an important fungal pathogen of small grain cereals that can also infect Arabidopsis thaliana. In Arabidopsis, jasmonic acid (JA) signalling involving JASMONATE RESISTANT 1 (JAR1), which synthesizes JA‐isoleucine, a signalling form of JA, promotes susceptibility to Fg. Here we show that Arabidopsis MYZUS PERSICAE‐INDUCED LIPASE 1 (MPL1), via its influence on limiting JA accumulation, restricts Fg infection. MPL1 expression was up‐regulated in response to Fg infection, and MPL1‐OE plants, which overexpress MPL1, exhibited enhanced resistance against Fg. In comparison, disease severity was higher on the mpl1 mutant than the wild type. JA content was lower in MPL1‐OE and higher in mpl1 than in the wild type, indicating that MPL1 limits JA accumulation. Pharmacological experiments confirmed the importance of MPL1‐determined restriction of JA accumulation on curtailment of Fg infection. Methyl‐JA application attenuated the MPL1‐OE‐conferred resistance, while the JA biosynthesis inhibitor ibuprofen enhanced resistance in mpl1. Also, the JA biosynthesis‐defective opr3 mutant was epistatic to mpl1, resulting in enhanced resistance in mpl1 opr3 plants. In comparison, JAR1 was not essential for the mpl1‐conferred susceptibility to Fg. Considering that methyl‐JA promotes Fg growth in culture, we suggest that in part MPL1 curtails disease by limiting the availability of a plant‐derived Fg growth‐promoting factor.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone.

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Molecular and physiological characterization of Arabidopsis–Colletotrichum gloeosporioides pathosystem

AbstractAnthracnose is a destructive disease that affects a wide range of crop plants especially in tropical and subtropical regions. Colletotrichum spp. are the major pathogens causing anthracnose. In this study, we collected and identified the pathogen from diseased samples of Stylosanthes, a major tropical forage crop. The ability of the pathogen to naturally infect Arabidopsis thaliana was examined. Sequence analysis of ITS, ACT, CHS, and GAPDH genes showed the pathogen to be Colletotrichum gloeosporioides sensu lato (s.l.), and this was supported further by morphological characterization of representative isolates. The disease symptoms and cellular infection process of aggressive isolates (DZ‐19 and HK‐04) and a weak isolate (CJ‐04) were compared. DZ‐19 and HK‐04 caused more severe disease symptoms on both young seedlings and adult plants of Col‐0 and Ws‐2 ecotypes compared to CJ‐04. Furthermore, the more aggressive isolates showed faster and earlier germination of conidia, formation of appressoria, and growth and development of hyphae during the infection. Genetic analysis of the defence response and expression profiling of defence marker genes demonstrated the involvement of MAP kinase, Ca2+‐dependent protein kinase, salicylic acid, ethylene, and jasmonic acid pathways in the resistance against anthracnose. These results suggest that the Arabidopsis–Colletotrichum gloeosporioides pathosystem should provide a valuable tool for exploring the resistance mechanisms against this pathogen.

A Chromosome-Scale Genome Assembly for the Fusarium oxysporum Strain Fo5176 To Establish a Model Arabidopsis-Fungal Pathosystem.

Plant pathogens cause widespread yield losses in agriculture. Understanding the drivers of plant-pathogen interactions requires decoding the molecular dialog leading to either resistance or disease. However, progress in deciphering pathogenicity genes has been severely hampered by suitable model systems and incomplete fungal genome assemblies. Here, we report a significant improvement of the assembly and annotation of the genome of the Fusarium oxysporum (Fo) strain Fo5176. Fo comprises a large number of serious plant pathogens on dozens of plant species with largely unresolved pathogenicity factors. The strain Fo5176 infects Arabidopsis thaliana and, hence, constitutes a highly promising model system. We use high-coverage Pacific Biosciences Sequel long-read and Hi-C sequencing data to assemble the genome into 19 chromosomes and a total genome size of 67.98 Mb. The genome has a N50 of 4 Mb and a 99.1% complete BUSCO score. Phylogenomic analyses based on single-copy orthologs clearly place the Fo5176 strain in the Fo f sp. conglutinans clade as expected. We generated RNAseq data from culture medium and plant infections to train gene predictions and identified ∼18,000 genes including ten effector genes known from other Fo clades. We show that Fo5176 is able to infect cabbage and Brussel sprouts of the Brassica oleracea, expanding the usefulness of the Fo5176 model pathosystem. Finally, we performed large-scale comparative genomics analyses comparing the Fo5176 to 103 additional Fo genomes to define core and accessory genomic regions. In conjunction with the molecular tool sets available for A. thaliana, the Fo5176 genome and annotation provides a crucial step toward the establishment of a highly promising pathosystem.

Double-Stranded RNA Pull-Down to Characterize Viral Replication Complexes in Plants.

Plant RNA viruses are obligate intracellular parasites that hijack specific cellular membranes to replicate their genomes in what are commonly known as viral replication complexes (VRC). These contain host- and virus-encoded proteins and viral RNA. Double-stranded RNA (dsRNA) is a mandatory intermediate of RNA replication and a hallmark feature of VRCs. We have recently developed a method to isolate viral dsRNA and its associated proteins through pull-down of an ectopically expressed dsRNA-binding protein (B2:GFP) from infected Arabidopsis thaliana plants. After mass spectrometry analysis to identify the dsRNA-associated proteins, resulting candidate proteins of interest are tagged with a red fluorescent protein and their subcellular localization in relation to VRCs is assessed by transient expression within leaves of B2:GFP-transgenic Nicotiana benthamiana plants. In this chapter we describe in detail these experimental procedures to allow investigators to characterize the replication complexes of their plant RNA virus of interest.

Dynamic Transcriptional Profiles of Arabidopsis thaliana Infected by Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is a negative-stranded RNA virus that infects hundreds of plant species, causing great economic loss. Infected Arabidopsis thaliana plants develop symptoms including chlorosis and wilt, which can lead to cell death. From 9 to 15 days after TSWV infection, symptoms progress through a three-stage process of appearance, severity, and death. In this study, deep sequencing technology was first used to explore gene expression in response to TSWV infection in model plant A. thaliana at different symptom development stages. We found that plant immune defense and protein degradation are induced by TSWV infection and that both inductions became stronger over time. The photosynthesis pathway was attenuated with TSWV infection. Cell wall metabolism had a large extent of downregulation while some genes were upregulated. These results illustrate the dynamic nature of TSWV infection in A. thaliana at the whole-transcriptome level. The link between biological processes and subpathway metabolism was further analyzed. Our study provides new insight into host regulatory networks and dynamic processes in response to TSWV infection.

Phytoplasma Transmission: Insect Rearing and Infection Protocols.

Phytoplasmas are obligate pathogens and thus they can be studied only in association with their plants or insect hosts. In this chapter, we present protocols for rearing some phytoplasma insect vectors, to obtain infected insects and plants under controlled environmental conditions. We focus on Euscelidius variegatus and Macrosteles quadripunctulatus that can infect Arabidopsis thaliana, and Hyalesthes obsoletus and Scaphoideus titanus, that can infect grapevine.

Phloem-Triggered Virus-Induced Gene Silencing Using a Recombinant Polerovirus.

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3′ non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.

Passion Fruit Chlorotic Mottle Virus: Molecular Characterization of a New Divergent Geminivirus in Brazil.

Brazil is one of the major passion fruit producers worldwide. Viral diseases are among the most important constraints for passion fruit production. Here we identify and characterize a new passion fruit infecting-virus belonging to the family Geminiviridae: passion fruit chlorotic mottle virus (PCMoV). PCMoV is a divergent geminivirus unlike previously characterized passion fruit-infecting geminiviruses that belonged to the genus Begomovirus. Among the presently known geminiviruses, it is most closely related to, and shares ~62% genome-wide identity with citrus chlorotic dwarf associated virus (CCDaV) and camelia chlorotic dwarf associated virus (CaCDaV). The 3743 nt PCMoV genome encodes a capsid protein (CP) and replication-associated protein (Rep) that respectively share 56 and 60% amino acid identity with those encoded by CaCDaV. The CPs of PCMoV, CCDaV, and CaCDaV cluster with those of begomovirus whereas their Reps with those of becurtoviruses. Hence, these viruses likely represent a lineage of recombinant begomo-like and becurto-like ancestral viruses. Furthermore, PCMoV, CCDaV, and CaCDaV genomes are ~12–30% larger than monopartite geminiviruses and this is primarily due to the encoded movement protein (MP; 891–921 nt) and this MP is most closely related to that encoded by the DNA-B component of bipartite begomoviruses. Hence, PCMoV, CCDaV, and CaCDaV lineage of viruses may represent molecules in an intermediary step in the evolution of bipartite begomoviruses (~5.3 kb) from monopartite geminiviruses (~2.7–3 kb). An infectious clone of PCMoV systemically infected Nicotiana benthamina, Arabidopsis thaliana, and Passiflora edulis.

The Plant-Dependent Life Cycle of Thecaphora thlaspeos: A Smut Fungus Adapted to Brassicaceae.

Smut fungi are globally distributed plant pathogens that infect agriculturally important crop plants such as maize or potato. To date, molecular studies on plant responses to smut fungi are challenging due to the genetic complexity of their host plants. Therefore, we set out to investigate the known smut fungus of Brassicaceae hosts, Thecaphora thlaspeos. T. thlaspeos infects different Brassicaceae plant species throughout Europe, including the perennial model plant Arabis alpina. In contrast to characterized smut fungi, mature and dry T. thlaspeos teliospores germinated only in the presence of a plant signal. An infectious filament emerges from the teliospore, which can proliferate as haploid filamentous cultures. Haploid filaments from opposite mating types mate, similar to sporidia of the model smut fungus Ustilago maydis. Consistently, the a and b mating locus genes are conserved. Infectious filaments can penetrate roots and aerial tissues of host plants, causing systemic colonization along the vasculature. Notably, we could show that T. thlaspeos also infects Arabidopsis thaliana. Exploiting the genetic resources of A. thaliana and Arabis alpina will allow us to characterize plant responses to smut infection in a comparative manner and, thereby, characterize factors for endophytic growth as well as smut fungi virulence in dicot plants.

Upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis thaliana.

A long intergenic noncoding RNA LINC - AP2 is upregulated and negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis. Plant vegetative growth and floral reproductive structure were severely retarded and distorted in Turnip crinkle virus (TCV)-infected Arabidopsis thaliana. Compared to mock-inoculated plants, the stamen filaments were shorter in flowers of TCV-infected plants. However, TCV-infected plants can still produce normal seeds through artificial pollination, indicating both its pollen and stigma were biologically functional. From our high-throughput RNA-Seq transcriptome analysis, a floral structure-related APETALA2 (AP2) gene was found to be downregulated and its neighboring long intergenic noncoding RNAs (lincRNA), At4NC069370 (named LINC-AP2 in this study), were upregulated significantly in TCV-infected plants. This LINC-AP2 was further confirmed for its existence using 5'RACE technology. LINC-AP2 overexpression (LINC-AP2 OE) transgenic Arabidopsis plants were generated to compare with TCV-infected WT plants. TCV-infected LINC-AP2 OE plants which contained lower AP2 gene expression displayed more severe symptoms (including floral structure distortion) and higher TCV-CP gene transcript and coat protein levels. Furthermore, compared to TCV-infected WT plants, TCV-infected ap2 mutant plants failed to open their flower buds and displayed more severe viral symptoms. In conclusion, upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with TCV infection in Arabidopsis.

Looking inside phytoplasma-infected sieve elements: A combined microscopy approach using Arabidopsis thaliana as a model plant

Phytoplasmas are phloem-inhabiting plant pathogens that affect over one thousand plant species, representing a severe threat to agriculture. The absence of an effective curative strategy and the economic importance of many affected crops make a priority of studying how plants respond to phytoplasma infection. Nevertheless, the study of phytoplasmas has been hindered by the extreme difficulty of culturing them in vitro and by impediments to natural host plant surveys such as low phytoplasma titre, long plant life cycle and poor knowledge of natural host-plant biology. Stating correspondence between macroscopic symptoms of phytoplasma infected Arabidopsis thaliana and those observed in natural host plants, over the last decade some authors have started to use this plant as a model for studying phytoplasma-plant interactions. Nevertheless, the morphological and ultrastructural modifications occurring in A. thaliana tissues following phytoplasma infection have never been described in detail. In this work, we adopted a combined-microscopy approach to verify if A. thaliana can be considered a reliable model for the study of phytoplasma-plant interactions at the microscopical level.The consistent presence of phytoplasma in infected phloem allowed detailed study of the infection process and the relationship established by phytoplasmas with different components of the sieve elements. In infected A. thaliana, phytoplasmas induced strong disturbances of host plant development that were mainly due to phloem disorganization and impairment. Light microscopy showed collapse, necrosis and hyperplasia of phloem cells. TEM observations of sieve elements identified two common plant-responses to phytoplasma infection: phloem protein agglutination and callose deposition.

Arabidopsis late blight: infection of a nonhost plant by Albugo laibachii enables full colonization by Phytophthora infestans.

SummaryThe oomycete pathogen Phytophthora infestans causes potato late blight, and as a potato and tomato specialist pathogen, is seemingly poorly adapted to infect plants outside the Solanaceae. Here, we report the unexpected finding that P. infestans can infect Arabidopsis thaliana when another oomycete pathogen, Albugo laibachii, has colonized the host plant. The behaviour and speed of P. infestans infection in Arabidopsis pre‐infected with A. laibachii resemble P. infestans infection of susceptible potato plants. Transcriptional profiling of P. infestans genes during infection revealed a significant overlap in the sets of secreted‐protein genes that are induced in P. infestans upon colonization of potato and susceptible Arabidopsis, suggesting major similarities in P. infestans gene expression dynamics on the two plant species. Furthermore, we found haustoria of A. laibachii and P. infestans within the same Arabidopsis cells. This Arabidopsis—A. laibachii—P. infestans tripartite interaction opens up various possibilities to dissect the molecular mechanisms of P. infestans infection and the processes occurring in co‐infected Arabidopsis cells.

Genome-wide transcriptomic analysis reveals correlation between higher WRKY61 expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana.

Turnip crinkle virus (TCV) is a carmovirus that infects many Arabidopsis ecotypes. Most studies mainly focused on discovery of resistance genes against TCV infection, and there is no Next Generation Sequencing based comparative genome wide transcriptome analysis reported. In this study, RNA-seq based transcriptome analysis revealed that 238 (155 up-regulated and 83 down-regulated) significant differentially expressed genes with at least 15-fold change were determined. Fifteen genes (including upregulated, unchanged and downregulated) were selected for RNA-seq data validation using quantitative real-time PCR, which showed consistencies between these two sets of data. GO enrichment analysis showed that numerous terms such as stress, immunity, defence and chemical stimulus were affected in TCV-infected plants. One putative plant defence related gene named WRKY61 was selected for further investigation. It showed that WRKY61 overexpression plants displayed reduced symptoms and less virus accumulation, as compared to wild type (WT) and WRKY61 deficient lines, suggesting that higher WRKY61 expression level reduced TCV viral accumulation. In conclusion, our transcriptome analysis showed that global gene expression was detected in TCV-infected Arabidopsis thaliana. WRKY61 gene was shown to be negatively correlated with TCV infection and viral symptoms, which may be connected to plant immunity pathways.

Arabidopsis Infection by Haploid or Diploid Strains of Ustilago maydis Reveals its Capacity as A Necrotrophic or Biotrophic Phytopathogen

The use of experimental patho-systems is a widely used strategy for understanding the pathogenic processes developed by many infectious agents. Ustilago maydis is a Basidiomycota fungus that naturally infects only maize and teozintle. We have established that under axenic conditions, either haploid or diploid strains of U. maydis can infect Arabidopsis thaliana, and in this work we analyzed the different behaviour of U. maydis haploid, diploid, and merodiploid strains in the mating genes during Arabidopsis infection. We determined the severity of infection, cell death, and the production of phytohormones and reactive oxygen species (ROS), occurring in the invaded host. Our results indicate that whereas U. maydis diploid and merodiploid strains behave as biotrophic parasites, the behaviour of the haploid strain is necrotrophic. We suggest that this difference resides in the fact that only the former two strains, but not the haploid one, are able to synthesize the master regulator of the biotrophic pathogenic behaviour, the bE/bW heterodimer encoded by the b genes of the two mating partners.

Functional and Genetic Analysis Identify a Role for Arabidopsis ARGONAUTE5 in Antiviral RNA Silencing.

RNA silencing functions as an antiviral defense through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. In turn, plant viruses have evolved strategies to counteract this defense mechanism, including the expression of suppressors of RNA silencing. Potato virus X (PVX) does not systemically infect Arabidopsis thaliana Columbia-0, but is able to do so effectively in mutants lacking at least two of the four Arabidopsis DCL proteins. PVX can also infect Arabidopsis ago2 mutants, albeit less effectively than double DCL mutants, suggesting that additional AGO proteins may mediate anti-viral defenses. Here we show, using functional assays, that all Arabidopsis AGO proteins have the potential to target PVX lacking its viral suppressor of RNA silencing (VSR), P25, but that only AGO2 and AGO5 are able to target wild-type PVX. However, P25 directly affects only a small subset of AGO proteins, and we present evidence indicating that its protective effect is mediated by precluding AGO proteins from accessing viral RNA, as well as by directly inhibiting the RNA silencing machinery. In agreement with functional assays, we show that Potexvirus infection induces AGO5 expression and that both AGO2 and AGO5 are required for full restriction of PVX infection in systemic tissues of Arabidopsis.

Molecular inversion probe: a new tool for highly specific detection of plant pathogens.

Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

Nondestructive Imaging of Plant-Parasitic Nematode Development and Host Response to Nematode Pathogenesis

The secluded lifestyle of endoparasitic plant nematodes hampers progress toward a comprehensive understanding of plant-nematode interactions. A novel technique that enables nondestructive, long-term observations of a wide range of live nematodes in planta is presented here. As proof of principle, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne chitwoodi were labeled fluorescently with PKH26 and used to infect Arabidopsis thaliana grown in microscopy rhizosphere chambers. Nematode behavior, development, and morphology were observed for the full duration of each parasite's life cycle by confocal microscopy for up to 27 days after inoculation. PKH26 accumulated in intestinal lipid droplets and had no negative effect on nematode infectivity. This technique enabled visualization of Meloidogyne gall formation, nematode oogenesis, and nematode morphological features, such as the metacorpus, vulva, spicules, and cuticle. Additionally, microscopy rhizosphere chambers were used to characterize plant organelle dynamics during M. chitwoodi infection. Peroxisome abundance strongly increased in early giant cells but showed a marked decrease at later stages of feeding site development, which suggests a modulation of plant peroxisomes by root-knot nematodes during the infection process. Taken together, this technique facilitates studies aimed at deciphering plant-nematode interactions at the cellular and subcellular level and enables unprecedented insights into nematode behavior in planta.