Characterization of a Host-Specific Toxic Activity Produced by Bipolaris cookei, Causal Agent of Target Leaf Spot of Sorghum.

Target leaf spot (TLS) of sorghum, caused by the necrotrophic fungus Bipolaris cookei, can cause severe yield loss in many parts of the world. We grew B. cookei in liquid culture and observed that the resulting culture filtrate (CF) was differentially toxic when infiltrated into the leaves of a population of 288 diverse sorghum lines. In this population, we found a significant correlation between high CF sensitivity and susceptibility to TLS. This suggests that the toxin produced in culture may play a role in the pathogenicity of B. cookei in the field. We demonstrated that the toxic activity is light sensitive and, surprisingly, insensitive to pronase, suggesting that it is not proteinaceous. We identified the two sorghum genetic loci most associated with the response to CF in this population. Screening seedlings with B. cookei CF could be a useful approach for prescreening germplasm for TLS resistance.

The maize ZmMIEL1 E3 ligase and ZmMYB83 transcription factor proteins interact and regulate the hypersensitive defence response.

The plant hypersensitive response (HR), a rapid cell death at the point of pathogenesis, is mediated by nucleotide-binding site, leucine-rich repeat (NLR) resistance proteins (R-proteins) that recognize the presence of specific pathogen-derived proteins. Rp1-D21 is an autoactive maize NLR R-protein that triggers HR spontaneously. We previously mapped loci associated with variation in the strength of HR induced by Rp1-D21. Here we identify the E3 ligase ZmMIEL1 as the causal gene at a chromosome 10 modifier locus. Transient ZmMIEL1 expression in Nicotiana benthamiana reduced HR induced by Rp1-D21, while suppression of ZmMIEL1 expression in maize carrying Rp1-D21 increased HR. ZmMIEL1 also suppressed HR induced by another autoactive NLR, the Arabidopsis R-protein RPM1D505V, in N. benthamiana. We demonstrated that ZmMIEL1 is a functional E3 ligase and that the effect of ZmMIEL1 was dependent on the proteasome but also that levels of Rp1-D21 and RPM1D505V were not reduced when coexpressed with ZmMIEL1 in the N. benthamiana system. By comparison to a similar system in Arabidopsis, we identify ZmMYB83 as a potential target of ZmMIEL1. Suppression of ZmMYB83 expression in maize lines carrying Rp1-D21 suppressed HR. Suppression of ZmMIEL1 expression caused an increase in ZmMYB83 transcript and protein levels in N. benthamiana and maize. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we demonstrated that ZmMIEL1 and ZmMYB83 physically interacted. Additionally, ZmMYB83 and ZmMIEL1 regulated the expression of a set of maize very long chain fatty acid (VLCFA) biosynthetic genes that may be involved in regulating HR.

Genome-wide association analysis of the strength of the MAMP-elicited defense response and resistance to target leaf spot in sorghum.

Plants have the capacity to respond to conserved molecular features known as microbe-associated molecular patterns (MAMPs). The goal of this work was to assess variation in the MAMP response in sorghum, to map loci associated with this variation, and to investigate possible connections with variation in quantitative disease resistance. Using an assay that measures the production of reactive oxygen species, we assessed variation in the MAMP response in a sorghum association mapping population known as the sorghum conversion population (SCP). We identified consistent variation for the response to chitin and flg22-an epitope of flagellin. We identified two SNP loci associated with variation in the flg22 response and one with the chitin response. We also assessed resistance to Target Leaf Spot (TLS) disease caused by the necrotrophic fungus Bipolaris cookei in the SCP. We identified one strong association on chromosome 5 near a previously characterized disease resistance gene. A moderately significant correlation was observed between stronger flg22 response and lower TLS resistance. Possible reasons for this are discussed.

Use of virus-induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize.

Plant disease resistance proteins (R-proteins) detect specific pathogen-derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1-D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1-D21-induced HR. Here we describe a system for characterizing genes mediating HR, using virus-induced gene silencing (VIGS) in a maize line carrying Rp1-D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R-proteins across several plant-pathogen systems. We confirmed that maize HSP90 was required for full Rp1-D21-induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1-D21-induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O-methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1-D21-induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.

The bHLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis.

KEY MESSAGE: ILR3 and PYE function in a regulatory network that modulates GLS accumulation under iron deficiency. The molecular processes involved in the cross talk between iron (Fe) homeostasis and other metabolic processes in plants are poorly understood. In Arabidopsis thaliana the transcription factor IAA-LEUCINE RESISTANT3 (ILR3) regulates iron deficiency response, aliphatic glucosinolate (GLS) biosynthesis and pathogen response. ILR3 is also known to interact with its homolog, POPEYE (PYE), which also plays a role in Fe response. However, little is known about how ILR3 regulates such diverse processes, particularly, via its interaction with PYE. Since GLS are produced as part of a defense mechanism against wounding pathogens, we examined pILR3::ß-GLUCURONIDASE expression and found that Fe deficiency enhances the wound-induced expression of ILR3 in roots and that ILR3 is induced in response to the wounding pathogen, sugarbeet root cyst nematode (Heterodera schachtii). We also examined the expression pattern of genes involved in Fe homeostasis and aliphatic GLS biosynthesis in pye-1, ilr3-2 and pye-1xilr3-2 (pxi) mutants and found that ILR3 and PYE differentially regulate the expression of genes involved these processes under Fe deficiency. We measured GLS levels and sugarbeet root cyst nematode infection rates under varying Fe conditions, and found that long-chain GLS levels are elevated in ilr3-2 and pxi mutants. This increase in long-chain GLS accumulation is correlated with elevated nematode resistance in ilr3-2 and pxi mutants in the absence of Fe. Our findings suggest that ILR3 and PYE function in a regulatory network that controls wounding pathogen response in plant roots by modulating GLS accumulation under iron deficiency.

Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::ß-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

Ironing out the issues: integrated approaches to understanding iron homeostasis in plants.

Plants initialize responses to environmental changes at all levels, from signaling to translation and beyond. Such is the case for fluctuations in the availability of iron (Fe), one of the most critical micronutrients for plants. The results of these responses are physiological and morphological changes that lead to increased iron uptake from the rhizosphere, and recycling and reallocation of Fe, which must be properly localized within specific cells and cellular compartment for use. The use of reductionist approaches, in combination with in vivo and in situ Fe localization tools, has been able to shed light on critical signaling molecules, transcriptional regulators, transporters and other proteins involved in Fe homeostasis. Recent advances in elemental distribution and speciation analysis now enable detection and measurement of Fe and other elements at resolutions never seen before. Moreover, increasing use of systems biology approaches provide a substantially broader perspective of how Fe availability affects processes at many levels. This review highlights the latest in vivo and in situ iron localization approaches and some of the recent advances in understanding mechanisms that control Fe translocation. A broad perspective of how Fe localization data might one day be integrated with large-scale data to create models for Fe homeostasis is presented.