Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.

BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

Suppressing chlorophyll degradation by silencing OsNYC3 improves rice resistance to Rhizoctonia solani, the causal agent of sheath blight.

Necrotrophic fungus Rhizoctonia solani Kühn (R. solani) causes serious diseases in many crops worldwide, including rice and maize sheath blight (ShB). Crop resistance to the fungus is a quantitative trait and resistance mechanism remains largely unknown, severely hindering the progress on developing resistant varieties. In this study, we found that resistant variety YSBR1 has apparently stronger ability to suppress the expansion of R. solani than susceptible Lemont in both field and growth chamber conditions. Comparison of transcriptomic profiles shows that the photosynthetic system including chlorophyll biosynthesis is highly suppressed by R. solani in Lemont but weakly in YSBR1. YSBR1 shows higher chlorophyll content than that of Lemont, and inducing chlorophyll degradation by dark treatment significantly reduces its resistance. Furthermore, three rice mutants and one maize mutant that carry impaired chlorophyll biosynthesis all display enhanced susceptibility to R. solani. Overexpression of OsNYC3, a chlorophyll degradation gene apparently induced expression by R. solani infection, significantly enhanced ShB susceptibility in a high-yield ShB-susceptible variety '9522'. However, silencing its transcription apparently improves ShB resistance without compromising agronomic traits or yield in field tests. Interestingly, altering chlorophyll content does not affect rice resistance to blight and blast diseases, caused by biotrophic and hemi-biotrophic pathogens, respectively. Our study reveals that chlorophyll plays an important role in ShB resistance and suppressing chlorophyll degradation induced by R. solani infection apparently improves rice ShB resistance. This discovery provides a novel target for developing resistant crop to necrotrophic fungus R. solani.

Jasmonate-Induced Defense Mechanisms in the Belowground Antagonistic Interaction Between Pythium arrhenomanes and Meloidogyne graminicola in Rice.

Next to their essential roles in plant growth and development, phytohormones play a central role in plant immunity against pathogens. In this study we studied the previously reported antagonism between the plant-pathogenic oomycete Pythium arrhenomanes and the root-knot nematode Meloidogyne graminicola, two root pathogens that co-occur in aerobic rice fields. In this manuscript, we investigated if the antagonism is related to imbalances in plant hormone levels, which could be involved in activation of plant defense. Hormone measurements and gene expression analyses showed that the jasmonate (JA) pathway is induced early upon P. arrhenomanes infection. Exogenous application of methyl-jasmonate (MeJA) on the plant confirmed that JA is needed for basal defense against both P. arrhenomanes and M. graminicola in rice. Whereas M. graminicola suppresses root JA levels to increase host susceptibility, Pythium inoculation boosts JA in a manner that prohibits JA repression by the nematode in double-inoculated plants. Exogenous MeJA supply phenocopied the defense-inducing capacity of Pythium against the root-knot nematode, whereas the antagonism was weakened in JA-insensitive mutants. Transcriptome analysis confirmed upregulation of JA biosynthesis and signaling genes upon P. arrhenomanes infection, and additionally revealed induction of genes involved in biosynthesis of diterpenoid phytoalexins, consistent with strong activation of the gene encoding the JA-inducible transcriptional regulator DITERPENOID PHYTOALEXIN FACTOR. Altogether, the here-reported data indicate an important role for JA-induced defense mechanisms in this antagonistic interaction. Next to that, our results provide evidence for induced expression of genes encoding ERF83, and related PR proteins, as well as auxin depletion in P. arrhenomanes infected rice roots, which potentially further contribute to the reduced nematode susceptibility seen in double-infected plants.

A single point mutation in the C-terminal extension of wheat Rubisco activase dramatically reduces ADP inhibition via enhanced ATP binding affinity.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca) is a AAA+ enzyme that uses ATP to remove inhibitors from the active site of Rubisco, the central carboxylation enzyme of photosynthesis. Rca α and ß isoforms exist in most higher plant species, with the α isoform being identical to the ß form but having an additional 25-45 amino acids at the Rca C terminus, known as the C-terminal extension (CTE). Rca is inhibited by ADP, and the extent of ADP sensitivity of the Rca complex can be modulated by the CTE of the α isoform, particularly in relation to a disulfide bond structure that is specifically reduced by the redox-regulatory enzyme thioredoxin-f. Here, we introduced single point mutations of Lys-428 in the CTE of Rca-α from wheat (Triticum aestivum) (TaRca2-α). Substitution of Lys-428 with Arg dramatically altered ADP inhibition, independently of thioredoxin-f regulation. We determined that the reduction in ADP inhibition in the K428R variant is not due to a change in ADP affinity, as the apparent constant for ADP binding was not altered by the K428R substitution. Rather, we observed that the K428R substitution strongly increased ATP substrate affinity and ATP-dependent catalytic velocity. These results suggest that the Lys-428 residue is involved in interacting with the γ-phosphate of ATP. Considering that nucleotide-dependent Rca activity regulates Rubisco and thus photosynthesis during fluctuating irradiance, the K428R substitution could potentially provide a mechanism for boosting the performance of wheat grown in the dynamic light environments of the field.

The energy sensor OsSnRK1a confers broad-spectrum disease resistance in rice.

Sucrose non-fermenting-1-related protein kinase-1 (SnRK1) belongs to a family of evolutionary conserved kinases with orthologs in all eukaryotes, ranging from yeasts (SnF1) to mammals (AMP-Activated kinase). These kinases sense energy deficits caused by nutrient limitation or stress and coordinate the required adaptations to maintain energy homeostasis and survival. In plants, SnRK1 is a global regulator of plant metabolism and is also involved in abiotic stress responses. Its role in the response to biotic stress, however, is only starting to be uncovered. Here we studied the effect of altered SnRK1a expression on growth and plant defense in rice. OsSnRK1a overexpression interfered with normal growth and development and increased resistance against both (hemi)biotrophic and necrotrophic pathogens, while OsSnRK1a silencing in RNAi lines increased susceptibility. OsSnRK1a overexpression positively affected the salicylic acid pathway and boosted the jasmonate-mediated defense response after inoculation with the blast fungus Pyricularia oryzae. Together these findings strongly suggest OsSnRK1a to be involved in plant basal immunity and favor a model whereby OsSnRK1a acts as a master switch that regulates growth-immunity trade-offs.

Target of rapamycin signaling orchestrates growth-defense trade-offs in plants.

Plant defense to microbial pathogens is often accompanied by significant growth inhibition. How plants merge immune system function with normal growth and development is still poorly understood. Here, we investigated the role of target of rapamycin (TOR), an evolutionary conserved serine/threonine kinase, in the plant defense response. We used rice as a model system and applied a combination of chemical, genetic, genomic and cell-based analyses. We demonstrate that ectopic expression of TOR and Raptor (regulatory-associated protein of mTOR), a protein previously demonstrated to interact with TOR in Arabidopsis, positively regulates growth and development in rice. Transcriptome analysis of rice cells treated with the TOR-specific inhibitor rapamycin revealed that TOR not only dictates transcriptional reprogramming of extensive gene sets involved in central and secondary metabolism, cell cycle and transcription, but also suppresses many defense-related genes. TOR overexpression lines displayed increased susceptibility to both bacterial and fungal pathogens, whereas plants with reduced TOR signaling displayed enhanced resistance. Finally, we found that TOR antagonizes the action of the classic defense hormones salicylic acid and jasmonic acid. Together, these results indicate that TOR acts as a molecular switch for the activation of cell proliferation and plant growth at the expense of cellular immunity.

Below-Ground Attack by the Root Knot Nematode Meloidogyne graminicola Predisposes Rice to Blast Disease.

Magnaporthe oryzae (rice blast) and the root-knot nematode Meloidogyne graminicola are causing two of the most important pathogenic diseases jeopardizing rice production. Here, we show that root-knot nematode infestation on rice roots leads to important above-ground changes in plant immunity gene expression, which is correlated with significantly enhanced susceptibility to blast disease. A detailed metabolic analysis of oxidative stress responses and hormonal balances demonstrates that the above-ground tissues have a disturbed oxidative stress level, with accumulation of H2O2, as well as hormonal disturbances. Moreover, double infection experiments on an oxidative stress mutant and an auxin-deficient rice line indicate that the accumulation of auxin in the above-ground tissue is at least partly responsible for the blast-promoting effect of root-knot nematode infection.

Lysine acetylation in mitochondria: From inventory to function.

Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field.

The DELLA Protein SLR1 Integrates and Amplifies Salicylic Acid- and Jasmonic Acid-Dependent Innate Immunity in Rice.

Gibberellins are a class of tetracyclic plant hormones that are well known to promote plant growth by inducing the degradation of a class of nuclear growth-repressing proteins, called DELLAs. In recent years, GA and DELLAs are also increasingly implicated in plant responses to pathogen attack, although our understanding of the underlying mechanisms is still limited, especially in monocotyledonous crop plants. Aiming to further decipher the molecular underpinnings of GA- and DELLA-modulated plant immunity, we studied the dynamics and impact of GA and DELLA during infection of the model crop rice (Oryza sativa) with four different pathogens exhibiting distinct lifestyles and infection strategies. Opposite to previous findings in Arabidopsis (Arabidopsis thaliana), our findings reveal a prominent role of the DELLA protein Slender Rice1 (SLR1) in the resistance toward (hemi)biotrophic but not necrotrophic rice pathogens. Moreover, contrary to the differential effect of DELLA on the archetypal defense hormones salicylic acid (SA) and jasmonic acid (JA) in Arabidopsis, we demonstrate that the resistance-promoting effect of SLR1 is due at least in part to its ability to boost both SA- and JA-mediated rice defenses. In a reciprocal manner, we found JA and SA treatment to interfere with GA metabolism and stabilize SLR1. Together, these findings favor a model whereby SLR1 acts as a positive regulator of hemibiotroph resistance in rice by integrating and amplifying SA- and JA-dependent defense signaling. Our results highlight the differences in hormone defense networking between rice and Arabidopsis and underscore the importance of GA and DELLA in molding disease outcomes.

Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) Alters Resistance to Bacterial and Fungal Pathogens.

Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus.

Comparative chemical screening and genetic analysis reveal tentoxin as a new virulence factor in Cochliobolus miyabeanus, the causal agent of brown spot disease on rice.

Brown spot disease, caused by Cochliobolus miyabeanus, is currently considered to be one of the most important yield reducers of rice (Oryza sativa L.). Despite its agricultural importance, little is known about the virulence mechanisms deployed by the fungus. Therefore, we set out to identify novel virulence factors with a role in disease development. This article reports, for the first time, the production of tentoxin by C. miyabeanus as a virulence factor during brown spot disease and the identification of the non-ribosomal protein synthetase (NRPS) CmNps3, responsible for tentoxin biosynthesis. We compared the chemical compounds produced by C. miyabeanus strains differing in virulence ability using ultra-high-performance liquid chromatography (UHPLC) coupled to high-resolution Orbitrap mass spectrometry (HRMS). The production of tentoxin by a highly virulent strain was revealed by principal component analysis of the detected ions and confirmed by UHPLC coupled to tandem-quadrupole mass spectrometry (MS/MS). The corresponding NRPS was identified by in silico genome analysis and confirmed by gene deletion. Infection tests with wild-type and Cmnps3 mutants showed that tentoxin acts as a virulence factor and is correlated with chlorosis development during the second phase of infection. Although rice has previously been classified as a tentoxin-insensitive plant species, our data demonstrate that tentoxin production by C. miyabeanus affects symptom development.

The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium.

Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21-amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

The role of thionins in rice defence against root pathogens.

Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in-depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root-knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differentially regulated by defence-related hormones, whereas all analysed genes were consistently down-regulated in M. graminicola-induced galls, at least until 7 days post-inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 revealed decreased susceptibility to M. graminicola infection and P. graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in defence against two of the most damaging root pathogens attacking rice.

Priming of wheat with the green leaf volatile Z-3-hexenyl acetate enhances defense against Fusarium graminearum but boosts deoxynivalenol production.

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.

Silicon induces resistance to the brown spot fungus Cochliobolus miyabeanus by preventing the pathogen from hijacking the rice ethylene pathway.

Although numerous studies have shown the ability of silicon (Si) to mitigate a wide variety of abiotic and biotic stresses, relatively little is known about the underlying mechanism(s). Here, we have investigated the role of hormone defense pathways in Si-induced resistance to the rice brown spot fungus Cochliobolus miyabeanus. To delineate the involvement of multiple hormone pathways, a multidisciplinary approach was pursued, combining exogenous hormone applications, pharmacological inhibitor experiments, time-resolved hormone measurements, and bioassays with hormone-deficient and/or -insensitive mutant lines. Contrary to other types of induced resistance, we found Si-induced brown spot resistance to function independently of the classic immune hormones salicylic acid and jasmonic acid. Our data also rule out a major role of the abscisic acid (ABA) and cytokinin pathways, but suggest that Si mounts resistance to C. miyabeanus by preventing the fungus from hijacking the rice ethylene (ET) machinery. Interestingly, rather than suppressing rice ET signaling per se, Si probably interferes with the production and/or action of fungal ET. Together our findings favor a scenario whereby Si induces brown spot resistance by disarming fungal ET and argue that impairment of pathogen virulence factors is a core resistance mechanism underpinning Si-induced plant immunity.

Phytohormone-mediated interkingdom signaling shapes the outcome of rice-Xanthomonas oryzae pv. oryzae interactions.

BACKGROUND: Small-molecule hormones are well known to play key roles in the plant immune signaling network that is activated upon pathogen perception. In contrast, little is known about whether phytohormones also directly influence microbial virulence, similar to what has been reported in animal systems. RESULTS: In this paper, we tested the hypothesis that hormones fulfill dual roles in plant-microbe interactions by orchestrating host immune responses, on the one hand, and modulating microbial virulence traits, on the other. Employing the rice-Xanthomonas oryzae pv. oryzae (Xoo) interaction as a model system, we show that Xoo uses the classic immune hormone salicylic acid (SA) as a trigger to activate its virulence-associated quorum sensing (QS) machinery. Despite repressing swimming motility, sodium salicylate (NaSA) induced production of the Diffusible Signal Factor (DSF) and Diffusible Factor (DF) QS signals, with resultant accumulation of xanthomonadin and extracellular polysaccharides. In contrast, abscisic acid (ABA), which favors infection by Xoo, had little impact on DF- and DSF-mediated QS, but promoted bacterial swimming via the LuxR solo protein OryR. Moreover, we found both DF and DSF to influence SA- and ABA-responsive gene expression in planta. CONCLUSIONS: Together our findings indicate that the rice SA and ABA signaling pathways cross-communicate with the Xoo DF and DSF QS systems and underscore the importance of bidirectional interkingdom signaling in molding plant-microbe interactions.

Primary metabolism plays a central role in moulding silicon-inducible brown spot resistance in rice.

Over recent decades, a multitude of studies have shown the ability of silicon (Si) to protect various plants against a range of microbial pathogens exhibiting different lifestyles and infection strategies. Despite this relative wealth of knowledge, an understanding of the action mechanism of Si is still in its infancy, which hinders its widespread application for agricultural purposes. In an attempt to further elucidate the molecular underpinnings of Si-induced disease resistance, we studied the transcriptome of control and Si-treated rice plants infected with the necrotrophic brown spot fungus Cochliobolus miyabeanus. Analysis of brown spot-infected control plants suggested that C. miyabeanus represses plant photosynthetic processes and nitrate reduction in order to trigger premature senescence and cause disease. In Si-treated plants, however, these pathogen-induced metabolic alterations are strongly impaired, suggesting that Si alleviates stress imposed by the pathogen. Interestingly, Si also significantly increased photorespiration rates in brown spot-infected plants. Although photorespiration is often considered as a wasteful process, recent studies have indicated that this metabolic bypass also enhances resistance during abiotic stress and pathogen attack by protecting the plant's photosynthetic machinery. In view of these findings, our results favour a scenario in which Si enhances brown spot resistance by counteracting C. miyabeanus-induced senescence and cell death via increased photorespiration. Moreover, our results shed light onto the mechanistic basis of Si-induced disease control and support the view that, in addition to activating plant immune responses, Si can also reduce disease severity by interfering with pathogen virulence strategies.

Making sense of hormone-mediated defense networking: from rice to Arabidopsis.

Phytohormones are not only essential for plant growth and development but also play central roles in triggering the plant immune signaling network. Historically, research aimed at elucidating the defense-associated role of hormones has tended to focus on the use of experimentally tractable dicot plants such as Arabidopsis thaliana. Emerging from these studies is a picture whereby complex crosstalk and induced hormonal changes mold plant health and disease, with outcomes largely dependent on the lifestyle and infection strategy of invading pathogens. However, recent studies in monocot plants are starting to provide additional important insights into the immune-regulatory roles of hormones, often revealing unique complexities. In this review, we address the latest discoveries dealing with hormone-mediated immunity in rice, one of the most important food crops and an excellent model for molecular genetic studies in monocots. Moreover, we highlight interactions between hormone signaling, rice defense and pathogen virulence, and discuss the differences and similarities with findings in Arabidopsis. Finally, we present a model for hormone defense networking in rice and describe how detailed knowledge of hormone crosstalk mechanisms can be used for engineering durable rice disease resistance.

Transcriptional behavior of EUL-related rice lectins toward important abiotic and biotic stresses.

The rice genome encodes several genes for putative carbohydrate-binding proteins belonging to the family of Euonymus related lectins (EULs). This lectin family was discovered recently and evidence shows that the expression of these proteins is subject to multiple environmental stresses. In this study, quantitative reverse transcription PCR (qRT-PCR) was conducted on rice seedlings exposed to various abiotic (150mM NaCl, 100mM mannitol, and 100µM abscisic acid (ABA)) and biotic (Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae) stresses to compare the transcriptional behavior of the EULs and a known stress related lectin Orysata belonging to the family of jacalin-related lectins. All EUL transcripts were strongly up-regulated after ABA and NaCl treatments in the roots whereas the overall expression level was generally lower and more variable in the shoots. Moreover, all abiotic stresses induced Orysata in both tissues except for mannitol treatment which failed to show an effect in the roots. Orysata also strongly accumulated after X. oryzae pv. oryzae infection, as were various D-type EUL lectins. In contrast, some of the EUL proteins, including OrysaEULS3, OrysaEULD1A and OrysaEULD2, as well as Orysata were significantly down-regulated upon M. oryzae attack, suggesting fungal manipulation of these genes. Collectively, our results clearly show that rice expresses multiple carbohydrate-binding proteins in response to a wide variety of abiotic and biotic stress conditions. We hypothesize that the Euonymus related proteins fulfill a prominent role in sensing and responding to multiple environmental cues.

Connecting growth and defense: the emerging roles of brassinosteroids and gibberellins in plant innate immunity.

Brassinosteroids (BRs) and gibberellins (GAs) are two groups of phytohormones that regulate many common developmental processes throughout the plant life cycle. Fueled by large-scale 'omics' technologies and the burgeoning field of plant computational biology, the past few years have witnessed paradigm-shifting advances in our understanding of how BRs and GA are perceived and their signals transduced. Accumulating evidence also implicates BR and GA in the coordination and integration of plant immune responses. Similarly to other growth regulators, BR and GA play ambiguous roles in molding pathological outcomes, the effects of which may depend not only on the pathogen's lifestyle and infection strategy, but also on specialized features of each interaction. Analysis of the underpinning molecular mechanisms points to a crucial role of GA-inhibiting DELLA proteins and the BR-regulated transcription factor BZR1. Acting at the interface of developmental and defense signaling, these proteins likely serve as central hubs for pathway crosstalk and signal integration, allowing appropriate modulation of plant growth and defense in response to various stimuli. In this review, we outline the latest discoveries dealing with BR and GA modulation of plant innate immunity and highlight interactions between BR and GA signaling, plant defense, and microbial virulence.