Karam B. Singh

Transcription factors in plant defense and stress responses

Transcriptional control of the expression of stress-responsive genes is a crucial part of the plant response to a range of abiotic and biotic stresses. Research carried out in the past few years has been productive in identifying transcription factors that are important for regulating plant responses to these stresses. These studies have also revealed some of the complexity and overlap in the responses to different stresses, and are likely to lead to new ways to enhance crop tolerance to disease and environmental stress.

Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement

Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea—desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.

The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations

SUMMARY The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.

Identification of Arabidopsis Ethylene-Responsive Element Binding Factors with Distinct Induction Kinetics after Pathogen Infection

Ethylene-responsive element binding factors (ERF) proteins are plant-specific transcription factors, many of which have been linked to stress responses. We have identified four Arabidopsis ERF genes whose expression was specifically induced by avirulent and virulent strains of the bacterial pathogen Pseudomonas syringae pv tomato, with overlapping but distinct induction kinetics. However, a delay in ERFmRNA accumulation after infection with the virulent strain was observed when compared with the avirulent strain. The induction ofERF gene expression in most cases preceded the mRNA accumulation of a basic chitinase gene, a potential downstream target for one or more of these ERFs. The expression of the ERFgenes was examined among different Arabidopsis tissues, in response to the signaling molecules ethylene, methyl jasmonate, and salicylic acid (SA), and in Arabidopsis mutants with decreased or enhanced susceptibility to pathogens, and significant differences were observed. For example, in seedlings, some of the ERF genes were not induced by SA in the wild-type but were SA responsive in thepad4-1 mutant, suggesting that PAD4-1, which acts upstream of SA accumulation, is also involved in repressing the SA-induced expression of specific ERF genes. The four ERF proteins were shown to contain transcriptional activation domains. These results suggest that transcriptional activation cascades involving ERF proteins may be important for plant defense to pathogen attack and that some ERF family members could be involved in the cross-talk between SA- and jasmonic acid-signaling pathways.

Aphid resistance in Medicago truncatula involves antixenosis and phloem-specific, inducible antibiosis, and maps to a single locus flanked by NBS-LRR resistance gene analogs

Aphids and related insects feed from a single cell type in plants: the phloem sieve element. Genetic resistance to Acyrthosiphon kondoi Shinji (bluegreen aphid or blue alfalfa aphid) has been identified in Medicago truncatula Gaert. (barrel medic) and backcrossed into susceptible cultivars. The status of M. truncatula as a model legume allows an in-depth study of defense against this aphid at physiological, biochemical, and molecular levels. In this study, two closely related resistant and susceptible genotypes were used to characterize the aphid-resistance phenotype. Resistance conditions antixenosis since migratory aphids were deterred from settling on resistant plants within 6 h of release, preferring to settle on susceptible plants. Analysis of feeding behavior revealed the trait affects A. kondoi at the level of the phloem sieve element. Aphid reproduction on excised shoots demonstrated that resistance requires an intact plant. Antibiosis against A. kondoi is enhanced by prior infestation, indicating induction of this phloem-specific defense. Resistance segregates as a single dominant gene, AKR (Acyrthosiphon kondoi resistance), in two mapping populations, which have been used to map the locus to a region flanked by resistance gene analogs predicted to encode the CC-NBS-LRR subfamily of resistance proteins. This work provides the basis for future molecular analysis of defense against phloem parasitism in a plant model system.

Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes

SummaryBiotic and abiotic stresses cause significant yield losses in legumes and can significantly affect their productivity. Biotechnology tools such as marker-assisted breeding, tissue culture, in vitro mutagenesis and genetic transformation can contribute to solve or reduce some of these constraints. However, only limited success has been achieved so far. The emergence of “omic” technologies and the establishment of model legume plants such as Medicago truncatula and Lotus japonicus are promising strategies for understanding the molecular genetic basis of stress resistance, which is an important bottleneck for molecular breeding. Understanding the mechanisms that regulate the expression of stress-related genes is a fundamental issue in plant biology and will be necessary for the genetic improvement of legumes. In this review, we describe the current status of biotechnology approaches in relation to biotic and abiotic stresses in legumes and how these useful tools could be used to improve resistance to important constraints affecting legume crops.

The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing multiple genes results in altered metabolic sensitivity to oxidative stress

Plant glutathione transferases (GSTs) are induced by diverse biotic and abiotic stimuli, and are important for protecting plants against oxidative damage. We have studied the primary transcriptional stress response of the entire Arabidopsis GST family to seven stresses, including both biotic and abiotic stimuli, with a focus on early changes in gene expression. Our results indicate that individual GST genes are highly specific in their induction patterns. Furthermore, we have been able to link individual GSTs to particular stress stimuli. Using RNAi, we successfully co-silenced a group of four phi GSTs that represent some of the most highly expressed GST genes. Despite a marked reduction in total phi GST protein levels, the transgenic plants showed no reduction in GST activity as measured using the model substrate 1-chloro-2,4-dinitrobenzene (CDNB), and appeared to have surprisingly robust physical phenotypes during stress. However, analysis of metabolite pools showed oxidation of the glutathione pool in the RNAi lines, and we observed alterations in carbon and nitrogen compounds following salicylic acid and hydrogen peroxide stress treatments, indicative of oxidative modification of primary metabolism. Thus, there appears to be a high degree of functional redundancy within the Arabidopsis GST family, with extensive disruption being required to reveal the roles of phi GSTs in protection against oxidative stress.

AtERF14, a Member of the ERF Family of Transcription Factors, Plays a Nonredundant Role in Plant Defense

We had previously shown that several transcription factors of the ethylene (ET) response factor (ERF) family were induced with different but overlapping kinetics following challenge of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae pv tomato DC3000 (avrRpt2). One of these genes, a transcriptional activator, AtERF14, was induced at the same time as ERF-target genes (ChiB, basic chitinase). To unravel the potential function of AtERF14 in regulating the plant defense response, we have analyzed gain- and loss-of-function mutants. We show here that AtERF14 has a prominent role in the plant defense response, since overexpression of AtERF14 had dramatic effects on both plant phenotype and defense gene expression and AtERF14 loss-of-function mutants showed impaired induction of defense genes following exogenous ET treatment and increased susceptibility to Fusarium oxysporum. Moreover, the expression of other ERF genes involved in defense and ET/jasmonic acid responses, such as ERF1 and AtERF2, depends on AtERF14 expression. A number of ERFs have been shown to function in the defense response through overexpression. However, the effect of loss of AtERF14 function on defense gene expression, pathogen resistance, and regulation of the expression of other ERF genes is unique thus far. These results suggest a unique role for AtERF14 in regulating the plant defense response.

Interactions between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences.

Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.

Mitochondrial complex II has a key role in mitochondrial-derived reactive oxygen species influence on plant stress gene regulation and defense

Mitochondria are both a source of ATP and a site of reactive oxygen species (ROS) production. However, there is little information on the sites of mitochondrial ROS (mROS) production or the biological role of such mROS in plants. We provide genetic proof that mitochondrial complex II (Complex II) of the electron transport chain contributes to localized mROS that regulates plant stress and defense responses. We identify an Arabidopsis mutant in the Complex II subunit, SDH1-1, through a screen for mutants lacking GSTF8 gene expression in response to salicylic acid (SA). GSTF8 is an early stress-responsive gene whose transcription is induced by biotic and abiotic stresses, and its expression is commonly used as a marker of early stress and defense responses. Transcriptional analysis of this mutant, disrupted in stress responses 1 (dsr1), showed that it had altered SA-mediated gene expression for specific downstream stress and defense genes, and it exhibited increased susceptibility to specific fungal and bacterial pathogens. The dsr1 mutant also showed significantly reduced succinate dehydrogenase activity. Using in vivo fluorescence assays, we demonstrated that root cell ROS production occurred primarily from mitochondria and was lower in the mutant in response to SA. In addition, leaf ROS production was lower in the mutant after avirulent bacterial infection. This mutation, in a conserved region of SDH1-1, is a unique plant mitochondrial mutant that exhibits phenotypes associated with lowered mROS production. It provides critical insights into Complex II function with implications for understanding Complex IIs role in mitochondrial diseases across eukaryotes.