Ron A. Salzman

Transcriptional Regulation of Sorghum Defense Determinants against a Phloem-Feeding Aphid

When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.

An Improved RNA Isolation Method for Plant Tissues Containing High Levels of Phenolic Compounds or Carbohydrates

Difficulties extracting high-quality RNA from recalcitrant plant tissues are often due to high levels of phenolics, carbohydrates, or other compounds that bind and/or co-precipitate with RNA. We describe here a method using soluble polyvinylpyrrolidone (PVP) and ethanol precipitation, which has been successful in several recalcitrant systems where other specialized RNA extraction methods failed to deliver suitable product. Using this method, RNA capable of reverse-transcription/PCR amplification and cDNA library construction was isolated from ripening grape berries, dry seeds of Albizia procera and radish, and leaf tissue of A. procera and Griffonia simplicifolia. This method is applicable to a variety of plant tissues.

NaCl Regulation of Plasma Membrane H+-ATPase Gene Expression in a Glycophyte and a Halophyte

NaCl regulation of plasma membrane H+-ATPase gene expression in the glycophyte tobacco (Nicotiana tabacum L. var Wisconsin 38) and the halophyte Atriplex nummularia L. was evaluated by comparison of organ-specific mRNA abundance using homologous cDNA probes encoding the ATPases of the respective plants. Accumulation of mRNA was induced by NaCl in fully expanded leaves and in roots but not in expanding leaves or stems. The NaCl responsiveness of the halophyte to accumulate plasma membrane H+-ATPase mRNA in roots was substantially greater than that of the glycophyte. Salt-induced transcript accumulation in A. nummularia roots was localized by in situ hybridization predominantly to the elongation zone, but mRNA levels also increased in the zone of differentiation. Increased message accumulation in A. nummularia roots could be detected within 8 h after NaCl (400 mM) treatment, and maximal levels were severalfold greater than in roots of untreated control plants. NaCl-induced plasma membrane H+-ATPase gene expression in expanded leaves and roots presumably indicates that these organs require increased H+-electrochemical potential gradients for the maintenance of plant ion homeostasis for salt adaptation. The greater capacity of the halophyte to induce plasma membrane H+-ATPase gene expression in response to NaCl may be a salt-tolerance determinant.

Transcriptional Profiling of Sorghum Induced by Methyl Jasmonate, Salicylic Acid, and Aminocyclopropane Carboxylic Acid Reveals Cooperative Regulation and Novel Gene Responses

We have conducted a large-scale study of gene expression in the C4 monocot sorghum (Sorghum bicolor) L. Moench cv BTx623 in response to the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene precursor aminocyclopropane carboxylic acid. Expression profiles were generated from seedling root and shoot tissue at 3 and 27 h, using a microarray containing 12,982 nonredundant elements. Data from 102 slides and quantitative reverse transcription-PCR data on mRNA abundance from 171 genes were collected and analyzed and are here made publicly available. Numerous gene clusters were identified in which expression was correlated with particular signaling compound and tissue combinations. Many genes previously implicated in defense responded to the treatments, including numerous pathogenesis-related genes and most members of the phenylpropanoid pathway, and several other genes that may represent novel activities or pathways. Genes of the octadecanoic acid pathway of jasmonic acid (JA) synthesis were induced by SA as well as by MeJA. The resulting hypothesis that increased SA could lead to increased endogenous JA production was confirmed by measurement of JA content. Comparison of responses to SA, MeJA, and combined SA+MeJA revealed patterns of one-way and mutual antagonisms, as well as synergistic effects on regulation of some genes. These experiments thus help further define the transcriptional results of cross talk between the SA and JA pathways and suggest that a subset of genes coregulated by SA and JA may comprise a uniquely evolved sector of plant signaling responsive cascades.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371–379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN‐free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN‐adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN‐induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L‐like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN‐degrading protease activity.

Arabidopsis vegetative storage protein is an anti-insect acid phosphatase

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the proteins secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor

Cowpea bruchid, when fed on a diet containing the soybean cysteine protease inhibitor soyacystatin N (scN), activates an array of counter‐defence genes to adapt to the negative effects of the inhibitor and regain its normal rate of feeding and development. A collection of 1920 cDNAs was obtained by differential subtraction with cDNAs prepared from guts of the 4th instar larvae of scN‐adapted (reared on scN‐containing diet) and scN‐unadapted (reared on regular scN‐free diet) cowpea bruchids. Subsequent expression profiling using DNA microarray and Northern blot analyses identified ninety‐four transcript species from this collection that are responsive to dietary scN. scN‐adapted insects induced genes encoding protein and carbohydrate digestive enzymes, probably to help meet their carbon and nitrogen requirements. Up‐regulation of antimicrobial and detoxification protein genes may represent a generalized defence response. Genes down‐regulated by scN reflected physiological adjustments of the cowpea bruchids to scN challenge. A large portion of the responsive genes, presumably involved in carrying out the counter‐defence response, were of unknown function. The full‐length cDNA of an scN‐inducible cathepsin B‐like cysteine protease was obtained. Its transcriptional response to scN during larval development contrasts with the pattern of the cathepsin L family, the major digestive enzymes. These results suggest cathepsin B‐like cysteine proteases may play a crucial role in cowpea bruchid adaptation to dietary scN.

Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor.

Upon challenge by the soybean cysteine protease inhibitor soyacystatin N (scN), cowpea bruchids reconfigure their major digestive cysteine proteases (CmCPs) in adaptation to the inhibitor and resume normal feeding and development. We have previously shown that CmCPB transcripts were 116.3‐fold more abundant in scN‐adapted bruchid guts than in unadapted guts, while CmCPA transcripts were only 2.5‐fold higher. In order to further elucidate the functional significance of this differential regulation, we expressed three CmCPA and one CmCPB isoforms (A9, A13, A16 and B1) using a bacterial expression system, and characterized their activities. In contrast to the precursors of CmCPAs (proCmCPAs), proCmCPB1 exhibited more efficient autocatalytic conversion from the latent proenzyme to its active mature protease form, and demonstrated higher intrinsic proteolytic activity. Among proCmCPAs, dependence on exogenous enzymatic processing varies: while maturation of proCmCPA13 and proCmCPA16 was impaired in the absence of external proteolytic activity, proCmCPA9 appeared to utilize a two‐step autoprocessing mechanism. Although all CmCPs are scN‐sensitive, scN was degraded by CmCPB1 when outnumbered by the protease, but scN remained intact in the presence of excessive CmCPA9. These results provide further evidence that differential expression of CmCPs under scN challenge brings about adaptation to the inhibitor. High induction of unique cysteine protease isoforms with superior autoprocessing and proteolytic efficacy represents a strategy cowpea bruchids use to cope with dietary scN.

Cowpea bruchid midgut transcriptome response to a soybean cystatin - Costs and benefits of counter-defence

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real‐time PCR (RT‐PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN‐responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN‐responsive genes were consistent in each larval stage, whereas others exhibited developmental stage‐specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up‐regulated by scN whereas structural, defence and stress‐related genes were largely down‐regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

Functional expression of an insect cathepsin B-like counter-defence protein.

Insects are capable of readjusting their digestive regimes in response to dietary challenge. Cowpea bruchids (Callosobruchus maculatus) strongly induce C. maculatus cathepsin B‐like cysteine protease 1 (CmCatB1) transcripts when fed diet containing a soybean cysteine protease inhibitor soyacystatin N (scN). CmCatB1 shares significant sequence similarity with cathepsin B‐like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1, but lacking a 70‐amino‐acid internal section. CmCatB1 and CmCatB2 probably resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. Further, this expression was only detectable in larvae, which is the developmental stage associated with food ingestion. The scN‐activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter‐defence against antinutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH‐dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E‐64 and the cathepsin B‐specific inhibitor CA‐074, verifying its cathepsin B‐like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. Together, we have provided functional evidence suggesting that CmCatB1 confers inhibitor‐insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors.