High-throughput phenotyping of nematode cysts.

The beet cyst nematode Heterodera schachtii is a plant pest responsible for crop loss on a global scale. Here, we introduce a high-throughput system based on computer vision that allows quantifying beet cyst nematode infestation and measuring phenotypic traits of cysts. After recording microscopic images of soil sample extracts in a standardized setting, an instance segmentation algorithm serves to detect nematode cysts in these images. In an evaluation using both ground truth samples with known cyst numbers and manually annotated images, the computer vision approach produced accurate nematode cyst counts, as well as accurate cyst segmentations. Based on such segmentations, cyst features could be computed that served to reveal phenotypical differences between nematode populations in different soils and in populations observed before and after the sugar beet planting period. The computer vision approach enables not only fast and precise cyst counting, but also phenotyping of cyst features under different conditions, providing the basis for high-throughput applications in agriculture and plant breeding research. Source code and annotated image data sets are freely available for scientific use.

Learning to Segment Fine Structures Under Image-Level Supervision With an Application to Nematode Segmentation.

Image segmentation models trained only with image-level labels have become increasingly popular as they require significantly less annotation effort than models trained with scribble, bounding box or pixel-wise annotations. While methods utilizing image-level labels achieve promising performance for the segmentation of larger-scale objects, they perform less well for the fine structures frequently encountered in biological images. In order to address this performance gap, we propose a deep network architecture based on two key principles, Global Weighted Pooling (GWP) and segmentation refinement by low-level image cues, that, together, make segmentation of fine structures possible. We apply our segmentation method to image datasets containing such fine structures, nematodes (worms + eggs) and nematode cysts immersed in organic debris objects, which is an application scenario encountered in automated soil sample screening. Supervised only with image-level labels, our approach achieves Dice coefficients of 79.72% and 58.51 % for nematode and nematode cyst segmentation, respectively.

High-throughput screening paradigms in ecotoxicity testing: Emerging prospects and ongoing challenges.

The rapidly increasing number of new production chemicals coupled with stringent implementation of global chemical management programs necessities a paradigm shift towards boarder uses of low-cost and high-throughput ecotoxicity testing strategies as well as deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity that can be used in effective risk assessment. The latter will require automated acquisition of biological data, new capabilities for big data analysis as well as computational simulations capable of translating new data into in vivo relevance. However, very few efforts have been so far devoted into the development of automated bioanalytical systems in ecotoxicology. This is in stark contrast to standardized and high-throughput chemical screening and prioritization routines found in modern drug discovery pipelines. As a result, the high-throughput and high-content data acquisition in ecotoxicology is still in its infancy with limited examples focused on cell-free and cell-based assays. In this work we outline recent developments and emerging prospects of high-throughput bioanalytical approaches in ecotoxicology that reach beyond in vitro biotests. We discuss future importance of automated quantitative data acquisition for cell-free, cell-based as well as developments in phytotoxicity and in vivo biotests utilizing small aquatic model organisms. We also discuss recent innovations such as organs-on-a-chip technologies and existing challenges for emerging high-throughput ecotoxicity testing strategies. Lastly, we provide seminal examples of the small number of successful high-throughput implementations that have been employed in prioritization of chemicals and accelerated environmental risk assessment.

Repetitive Transcranial Magnetic Stimulation For The Treatment of Chronic Tinnitus: A Preliminary Study of The Influence of Traumatic Brain Injury on Treatment Response.

This study aims to test whether the efficacy of repetitive Transcranial Magnetic Stimulation (rTMS) differs between patients who developed tinnitus following a traumatic brain injury (TBI), and those without a history of TBI. This was a parallel pilot, open-label, non-randomized, clinical trial to compare the efficacy of low frequency rTMS on tinnitus symptoms in patients with and without a TBI history. Patients with moderate to severe tinnitus symptoms based on the Tinnitus Handicap Inventory (THI) and the Tinnitus Functional Index (TFI) were enrolled in the study. Validated questionnaires (THI and TFI) were used to quantify the severity of tinnitus symptoms and hearing impairment (Hearing Handicap Index - HHI) before and after ten sessions of rTMS of the left primary auditory cortex. Hearing threshold levels as well as speech reception and speech discrimination thresholds were also compared. The number of patients who experienced a reduction in their subjective tinnitus symptoms was greater and sustained longer in patients without a history of TBI. The same was seen with subjective symptoms of hearing impairment. In conclusion, our preliminary results suggest tinnitus patients without a history of TBI respond better to low frequency rTMS than those with a history of TBI, suggesting that treatments could be more effective if tailored to tinnitus etiology.

Low-glutathione mutants are impaired in growth but do not show an increased sensitivity to moderate water deficit.

Glutathione is considered a key metabolite for stress defense and elevated levels have frequently been proposed to positively influence stress tolerance. To investigate whether glutathione affects plant performance and the drought tolerance of plants, wild-type Arabidopsis plants and an allelic series of five mutants (rax1, pad2, cad2, nrc1, and zir1) with reduced glutathione contents between 21 and 63% compared to wild-type glutathione content were phenotypically characterized for their shoot growth under control and water-limiting conditions using a shoot phenotyping platform. Under non-stress conditions the zir1 mutant with only 21% glutathione showed a pronounced dwarf phenotype. All other mutants with intermediate glutathione contents up to 62% in contrast showed consistently slightly smaller shoots than the wild-type. Moderate drought stress imposed through water withdrawal until shoot growth ceased showed that wild-type plants and all mutants responded similarly in terms of chlorophyll fluorescence and growth retardation. These results lead to the conclusion that glutathione is important for general plant performance but that the glutathione content does not affect tolerance to moderate drought conditions typically experienced by crops in the field.

Instance Segmentation of Nematode Cysts in Microscopic Images of Soil Samples.

Nematodes are plant parasites that cause damage to crops. In order to quantify nematode infestation based on soil samples, we propose an instance segmentation method that will serve as the basis of automatic quantitative analysis. We consider light microscopic images of cluttered object collections as they occur in realistic soil samples. We introduce an algorithm, LMBI (Local Maximum of Boundary Intensity) to propose instance segmentation candidates. In a second step, a SVM classifier separates the nematode cysts among the candidates from soil particles. On a data set of soil sample images, the LMBI detector achieves near-optimal recall with a limited number of candidate segmentations, and the combined detector/classifier achieves recall and precision of 0.7. The pipeline only requires simple dot annotations and ≈moderately sized training data, which enables quick annotating and training in laboratory applications.

Phenotype of Arabidopsis thaliana semi-dwarfs with deep roots and high growth rates under water-limiting conditions is independent of the GA5 loss-of-function alleles.

BACKGROUND AND AIMS: The occurrence of Arabidopsis thaliana semi-dwarf accessions carrying inactive alleles at the gibberellin (GA) biosynthesis GA5 locus has raised the question whether there are pleiotropic effects on other traits at the root level, such as rooting depth. In addition, it is unknown whether semi-dwarfism in arabidopsis confers a growth advantage under water-limiting conditions compared with wild-type plants. The aim of this research was therefore to investigate whether semi-dwarfism has a pleiotropic effect in the root system and also whether semi-dwarfs might be more tolerant of water-limiting conditions. METHODS: The root systems of different arabidopsis semi-dwarfs and GA biosynthesis mutants were phenotyped in vitro using the GROWSCREEN-ROOT image-based software. Semi-dwarfs were phenotyped together with tall, near-related accessions. In addition, root phenotypes were investigated in soil-filled rhizotrons. Rosette growth trajectories were analysed with the GROWSCREEN-FLUORO setup based on non-invasive imaging. KEY RESULTS: Mutations in the early steps of the GA biosynthesis pathway led to a reduction in shoot as well as root size. Depending on the genetic background, mutations at the GA5 locus yielded phenotypes characterized by decreased root length in comparison with related wild-type ones. The semi-dwarf accession Pak-3 showed the deepest root system both in vitro and in soil cultivation experiments; this comparatively deep root system, however, was independent of the ga5 loss-of-function allele, as shown by co-segregation analysis. When the accessions were grown under water-limiting conditions, semi-dwarf accessions with high growth rates were identified. CONCLUSIONS: The observed diversity in root system growth and architecture occurs independently of semi-dwarf phenotypes, and is probably linked to a genetic background effect. The results show that there are no clear advantages of semi-dwarfism at low water availability in arabidopsis.

Shoot and root phenotyping of the barley mutant kcs6 (3-ketoacyl-CoA synthase6) depleted in epicuticular waxes under water limitation.

Aerial parts of plants are separated from the environment by a cuticle which functions in protection against desiccation and pathogen attack. Recently, we reported on a barley mutant with defect in the 3-KETOACYL-CoA-SYNTHASE (HvKCS6) gene, resulting in reduced coverage of the cuticle with epicuticular waxes. Spores of adapted and non-adapted powdery mildew fungi germinated less frequently on mutant leaves possibly because plant derived signals are missing. We used a shoot and root phenotyping facility to test whether depletion in epicuticular waxes negatively impacts plant performance under water-limiting conditions. While shoots of mutant plants grew slower at well-watered conditions than wild-type plants, they showed an equal or slightly better growth rate at water limitation. Also for roots, differences between mutant and parental line were less prominent at water-limiting as compared to well-watered conditions. Our results challenge the intuitive belief that reduced epicuticular wax might become a drawback at water limitation.

Magnetic resonance imaging of sugar beet taproots in soil reveals growth reduction and morphological changes during foliar Cercospora beticola infestation.

Cercospora leaf spot (CLS) infection can cause severe yield loss in sugar beet. Introduction of Cercospora-resistant varieties in breeding programmes is important for plant protection to reduce both fungicide applications and the risk of the development of fungal resistance. However, in vivo monitoring of the sugar-containing taproots at early stages of foliar symptoms and the characterization of the temporal development of disease progression has proven difficult. Non-invasive magnetic resonance imaging (MRI) measurements were conducted to quantify taproot development of genotypes with high (HS) and low (LS) levels of susceptibility after foliar Cercospora inoculation. Fourteen days post-inoculation (dpi) the ratio of infected leaf area was still low (~7%) in both the HS and LS genotypes. However, during this period, the volumetric growth of the taproot had already started to decrease. Additionally, inoculated plants showed a reduction of the increase in width of inner cambial rings while the width of outer rings increased slightly compared with non-inoculated plants. This response partly compensated for the reduced development of inner rings that had a vascular connection with Cercospora-inoculated leaves. Hence, alterations in taproot anatomical features such as volume and cambial ring development can be non-invasively detected already at 14 dpi, providing information on the early impact of the infection on whole-plant performance. All these findings show that MRI is a suitable tool to identify promising candidate parent lines with improved resistance to Cercospora, for example with comparatively lower taproot growth reduction at early stages of canopy infection, for future introduction into breeing programmes.

HyperART: non-invasive quantification of leaf traits using hyperspectral absorption-reflectance-transmittance imaging.

BACKGROUND: Combined assessment of leaf reflectance and transmittance is currently limited to spot (point) measurements. This study introduces a tailor-made hyperspectral absorption-reflectance-transmittance imaging (HyperART) system, yielding a non-invasive determination of both reflectance and transmittance of the whole leaf. We addressed its applicability for analysing plant traits, i.e. assessing Cercospora beticola disease severity or leaf chlorophyll content. To test the accuracy of the obtained data, these were compared with reflectance and transmittance measurements of selected leaves acquired by the point spectroradiometer ASD FieldSpec, equipped with the FluoWat device. RESULTS: The working principle of the HyperART system relies on the upward redirection of transmitted and reflected light (range of 400 to 2500 nm) of a plant sample towards two line scanners. By using both the reflectance and transmittance image, an image of leaf absorption can be calculated. The comparison with the dynamically high-resolution ASD FieldSpec data showed good correlation, underlying the accuracy of the HyperART system. Our experiments showed that variation in both leaf chlorophyll content of four different crop species, due to different fertilization regimes during growth, and fungal symptoms on sugar beet leaves could be accurately estimated and monitored. The use of leaf reflectance and transmittance, as well as their sum (by which the non-absorbed radiation is calculated) obtained by the HyperART system gave considerably improved results in classification of Cercospora leaf spot disease and determination of chlorophyll content. CONCLUSIONS: The HyperART system offers the possibility for non-invasive and accurate mapping of leaf transmittance and absorption, significantly expanding the applicability of reflectance, based on mapping spectroscopy, in plant sciences. Therefore, the HyperART system may be readily employed for non-invasive determination of the spatio-temporal dynamics of various plant properties.

Evolutionary conserved function of barley and Arabidopsis 3-KETOACYL-CoA SYNTHASES in providing wax signals for germination of powdery mildew fungi.

For plant pathogenic fungi, such as powdery mildews, that survive only on a limited number of host plant species, it is a matter of vital importance that their spores sense that they landed on the right spot to initiate germination as quickly as possible. We investigated a barley (Hordeum vulgare) mutant with reduced epicuticular leaf waxes on which spores of adapted and nonadapted powdery mildew fungi showed reduced germination. The barley gene responsible for the mutant wax phenotype was cloned in a forward genetic screen and identified to encode a 3-KETOACYL-CoA SYNTHASE (HvKCS6), a protein participating in fatty acid elongation and required for synthesis of epicuticular waxes. Gas chromatography-mass spectrometry analysis revealed that the mutant has significantly fewer aliphatic wax constituents with a chain length above C-24. Complementation of the mutant restored wild-type wax and overcame germination penalty, indicating that wax constituents less present on the mutant are a crucial clue for spore germination. Investigation of Arabidopsis (Arabidopsis thaliana) transgenic plants with sense silencing of Arabidopsis REQUIRED FOR CUTICULAR WAX PRODUCTION1, the HvKCS6 ortholog, revealed the same germination phenotype against adapted and nonadapted powdery mildew fungi. Our findings hint to an evolutionary conserved mechanism for sensing of plant surfaces among distantly related powdery mildews that is based on KCS6-derived wax components. Perception of such a signal must have been evolved before the monocot-dicot split took place approximately 150 million years ago.

Regulation of growth by the trehalose pathway: relationship to temperature and sucrose.

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.

Non-invasive approaches for phenotyping of enhanced performance traits in bean.

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene-environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure-function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Cyclic monoterpene mediated modulations of Arabidopsis thaliana phenotype: effects on the cytoskeleton and on the expression of selected genes.

Monoterpenes at high atmospheric concentrations are strong growth inhibitors in allelopathic interactions. Effects depend on dose, molecular structure of the monoterpene and on the species of the receiver plant. Stomata are among the first targets affected by camphor and menthol. Previously, it could be demonstrated that the compounds induce swelling of the protoplasts, prevent stomatal closure and enhance transpiration. In this study, we show that the block of stomatal closure is accompanied by changes to the cytoskeleton, which has a direct role in stomatal movements. Although MPK3 (MAP3 kinase) and ABF4 gene expressions are induced within six hours, stomatal closure is prevented. In contrast to ABF4, ABF2 (both transcription factors) is not induced. MPK3 and ABF4 both encode for proteins involved in the process of stomatal closure. The expression of PEPCase, an enzyme important for stomatal opening, is down regulated. The leaves develop stress symptoms, mirrored by transient changes in the expression profile of additional genes: lipoxygenase 2 (LOX2), CER5, CER6 (both important for wax production) and RD29B (an ABA inducible stress protein). Non-invasive methods showed a fast response of the plant to camphor fumigations both in a rapid decrease of the quantum yield and in the relative growth rate. Repeated exposures to the monoterpenes resulted finally in growth reduction and a stress related change in the phenotype. It is proposed that high concentrations or repeated exposure to monoterpenes led to irreversible damages, whereas low concentrations or short-term fumigations may have the potential to strengthen the plant fitness.

Simultaneous phenotyping of leaf growth and chlorophyll fluorescence via GROWSCREEN FLUORO allows detection of stress tolerance in Arabidopsis thaliana and other rosette plants.

Stress caused by environmental factors evokes dynamic changes in plant phenotypes. In this study, we deciphered simultaneously the reaction of plant growth and chlorophyll fluorescence related parameters using a novel approach which combines existing imaging technologies (GROWSCREEN FLUORO). Three different abiotic stress situations were investigated demonstrating the benefit of this approach to distinguish between effects related to (1) growth, (2) chlorophyll-fluorescence, or (3) both of these aspects of the phenotype. In a drought stress experiment with more than 500 plants, poly(ADP-ribose) polymerase (PARP) deficient lines of Arabidopsis thaliana (L.) Heynh showed increased relative growth rates (RGR) compared with C24 wild-type plants. In chilling stress, growth of PARP and C24 lines decreased rapidly, followed by a decrease in Fv/Fm. Here, PARP-plants showed a more pronounced decrease of Fv/Fm than C24, which can be interpreted as a more efficient strategy for survival in mild chilling stress. Finally, the reaction of Nicotiana tabacum L. to altered spectral composition of the intercepted light was monitored as an example of a moderate stress situation that affects chlorophyll-fluorescence related, but not growth-related parameters. The examples investigated in this study show the capacity for improved plant phenotyping based on an automated and simultaneous evaluation of growth and photosynthesis at high throughput.

Barley Rom1 antagonizes Rar1 function in Magnaporthe oryzae-infected leaves by enhancing epidermal and diminishing mesophyll defence.

* Barley (Hordeum vulgare) is a host for Blumeria graminis f. sp. hordei (Bgh), which causes powdery mildew, and for the rice blast pathogen Magnaporthe oryzae. It has previously been shown that Rar1, initially identified in a mutational screen as being required for Mla12-specified Bgh-resistance, also controlled pathogenic growth of M. oryzae in barley. Here, we tested whether the rom1 mutation (restoration of Mla12-specified resistance), which restored resistance against Bgh in a susceptible rar1-2 genetic background, also influences the interaction between barley and M. oryzae. * Disease severity after infection with M. oryzae was analysed on rar1-2 mutants and rar1-2 rom1 double mutants. Microscopy and northern analysis were used to gain insight into cellular and molecular events. * On rar1-2 rom1 double mutant plants, the number of M. oryzae disease lesions was increased in comparison to the wild-type and the rar1-2 mutant which correlated with augmented epidermal penetration. However, a decrease in the lesion diameter, apparently conditioned in the mesophyll, was also observed. * These results highlight the impact of Rom1 in basal defence of barley against different pathogens. Importantly, a tissue-specific function for Rom1 with contrasting effects on epidermal and mesophyll defence was demonstrated.

The barley mutant emr1 exhibits restored resistance against Magnaporthe oryzae in the hypersusceptible mlo-genetic background.

Barley plants having wild-type or mutant alleles at the MLO locus show opposite responses to infection with different pathogens, i.e. plants homozygous for mutant alleles (mlo) are resistant to powdery mildew but hypersusceptible to the rice blast fungus Magnaporthe oryzae and vice versa for plants with at least one wild-type MLO-allele. A mutational analysis was performed in the mlo-genetic background aimed at identifying of individuals with restored resistance against M. oryzae. Here, we describe the barley enhanced Magnaporthe resistance (emr1) mutant which showed restored resistance against blast in the absence of wild-type MLO. The emr1 mutant could be classified as a loss of function mutant. It could be excluded that resistance of emr1 is a back-mutation at the mlo-locus, because emr1 retained resistance against Bgh. The mutant did not display generally increased resistance as was evidenced by infection with either brown rust or net blotch pathogens. Additionally, resistance in emr1 was not associated with constitutively activated defence as confirmed by monitoring PR-gene transcript accumulation. Microscopic analysis showed that resistance of the emr1 mutant against M. oryzae was correlated with blocked penetration in epidermal cells and a concomitantly reduced progression into the mesophyll. These findings are reminiscent of the defence phenotypes against M. oryzae previously described for wild-type barley MLO genotypes. Therefore, it is tempting to speculate that resistance in the emr1 mutant was regained by the knockdown of putative suppressor element(s) acting in the defence scenario against M. oryzae, which diminish resistance only in mlo but not in MLO genotypes.

The Barley Mutant emr1 was Identified in a Mutational Screen for Resistance Against Magnaporthe oryzae.

Barley plants can be colonized by the fungus Magnaporthe oryzae, a pathogen initially known from rice plant cultivation. A mutational screen was performed in the barley mlo-genetic background which is, in comparison to wild-type MLO-genotypes, hypersusceptible against this fungus. This led to the identification of a mutant, referred to as emr1 (enhanced Magnaporthe resistance), that showed partially restored resistance. Disease symptoms on leaves of emr1 were significantly less severe than on mlo5-genotypes but still more than on wt MLO-barley plants.Segregation analysis showed that emr1 was inherited as a single recessive trait. Insight into the mode of action of emr1-dependent resistance against M. oryzae was gained by microscopic analysis. The results of these experiments revealed that mutant emr1 blocked penetration by M. oryzae by the formation of effective papillae in approximately half of all incidences. At about 30% of the interaction sites fungal growth was arrested effectively by an HR in the epidermal cell. Only a low frequency of fungal infection sites proceed into the mesophyll where fungal invasion resulted in the onset of a hypersensitive response (HR)-like cell death. Here, we report further evidence that barley shows a mesophyll HR in response to colonisation by M. oryzae. The possibility that the fungus turns this ostensible defence reaction to its own advantage and profits from the dead host tissue by switching to a necrotrophic lifestyle is discussed.

Competence of roots for race-specific resistance and the induction of acquired resistance against Magnaporthe oryzae.

SUMMARY Generally, Magnaporthe oryzae, the causal agent of rice blast disease, is considered to be a typical leaf-infecting plant pathogenic fungus. However, it was recently reported that M. oryzae shares many characteristics in common with root-infecting pathogens and indeed was able to infect roots. Here, we report on studies testing for the capacity of roots of rice and barley to resist infections with M. oryzae. We established that roots of rice plants were colonized by M. oryzae in a manner which is different from the gene-for-gene specificity seen in leaves for the same genotypes. Furthermore, treatment of rice seedlings with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a chemical that protects leaves effectively against blast by conditioning acquired resistance, was not able to prevent colonization of roots by M. oryzae although a reduction in disease levels was observed. Moreover, BTH was not able to protect barley roots against infection with M. oryzae. Taken together, our results suggest that although roots show intrinsic variation in their ability to resist colonization by M. oryzae, neither gene-for-gene incompatibility nor aquired resistance are as effective at blocking the pathogen as they are in leaves.

Acquired resistance functions in mlo barley, which is hypersusceptible to Magnaporthe grisea.

Barley plants carrying a mutation in the Mlo (barley [Hordeum vulgare L.] cultivar Ingrid) locus conferring a durable resistance against powdery mildew are hypersusceptible to the rice blast fungus Magnaporthe grisea. It has been speculated that a functional Mlo gene is required for the expression of basic pathogen resistance and that the loss of Mlo function mediating powdery mildew resistance is an exception for this particular disease. Here, we report that the onset of acquired resistance (AR) after chemical as well as biological treatments is sufficient to overcome the hypersusceptible phenotype of backcross line BCIngridmlo5 (mlo) barley plants against M. grisea. Moreover, even barley plants bearing a functional Mlo gene and thus showing a moderate infection phenotype against rice blast exhibit a further enhanced resistance after induction of AR. Cytological investigations reveal that acquired resistance in mlo genotypes is manifested by the restoration of the ability to form an effective papilla at sites of attempted penetration, similarly to wild-type Mlo plants. In addition, the rate of effective papillae formation in Mlo plants was further enhanced after the onset of AR. These results demonstrate that treatments leading to the AR state in barley function independently of the Mlo/mlo phenotype and suggest that the Mlo protein is not a component of the AR signaling network. Moreover, it seems that only concomitant action of Mlo together with AR permits high level resistance in barley against blast. Higher steady state levels of PR1 and barley chemically induced mRNA correlate with higher disease severity rather than with the degree of resistance observed in this particular interaction.