John P. Klingler

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.

Involvement of the octadecanoid pathway in bluegreen aphid resistance in Medicago truncatula

Aphids are major insect pests of plants that feed directly from the phloem. We used the model legume Medicago truncatula Gaert. (barrel medic) to elucidate host resistance to aphids and identified a single dominant gene which confers resistance to Acyrthosiphon kondoi Shinji (bluegreen aphid). To understand how this gene conditions resistance to bluegreen aphid, transcription profiling of 23 defense-related genes representing various signaling pathways was undertaken using a pair of near-isogenic lines that are susceptible or resistant to bluegreen aphid. All salicylic acid- and ethylene-responsive genes tested were induced by bluegreen aphid in resistant and susceptible plants, although there were some differences in the magnitude and kinetics of the induction. In contrast, 10 of 13 genes associated with the octadecanoid pathway were induced exclusively in the resistant plants following bluegreen aphid infestation. These results are in contrast to plant-pathogen interactions where similar sets of defense genes typically are induced in compatible interactions, but to a lesser degree and later than in incompatible interactions. Treatment of susceptible plants with methyl jasmonate reduced bluegreen aphid infestation but not to the same levels as the resistant line. Together, these results strongly suggest that the octadecanoid pathway is important for this naturally derived aphid resistance trait.

Phloem specific aphid resistance in Cucumis melo line AR 5 : effects on feeding behaviour and performance of Aphis gossypii

The feeding behaviour, excretion rate, and life history traits of the cotton‐melon aphid, Aphis gossypii (Glover) (Homoptera, Aphididae), were measured on a resistant melon, Cucumis melo L., breeding line, AR 5. The site of resistance detection by the aphids was determined using the electrical penetration graph (EPG) technique. EPG recordings showed that resistance is expressed within the host plant, rather than on its surface, because the time to first stylet penetration was not significantly different between AR 5 and the closely related susceptible breeding line, PMR 5. EPG patterns associated with stylet pathway activities of the aphids were not significantly different between the resistant and susceptible lines. Significant behavioural differences were observed only after stylets contacted phloem sieve elements. On AR 5, the duration of salivation after sieve element puncture (waveform E1) was significantly longer, and the number of aphids showing phloem sap ingestion (waveform E2) was significantly reduced. We conclude that the resistance mechanism producing the effects seen in this study acts within the phloem sieve elements. Monitoring of excretion rates on the two genotypes showed that aphid feeding was delayed and greatly reduced on the resistant genotype. Comparisons of aphid life history traits and population development between host plant genotypes showed that the effects of resistance act throughout aphid development and are highly effective at slowing down population increase.

Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance

Abstract.Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed.

Characterization of Pea Aphid Resistance in Medicago truncatula

To achieve a thorough understanding of plant-aphid interactions, it is necessary to investigate in detail both the plant and insect side of the interaction. The pea aphid (PA; Acyrthosiphon pisum) has been selected by an international consortium as the model species for genetics and genomics studies, and the model legume Medicago truncatula is a host of this aphid. In this study, we identified resistance to PA in a M. truncatula line, ‘Jester’, with well-characterized resistance to a closely related aphid, the bluegreen aphid (BGA; Acyrthosiphon kondoi). The biology of resistance to the two aphid species shared similarity, with resistance in both cases occurring at the level of the phloem, requiring an intact plant and involving a combination of antixenosis, antibiosis, and plant tolerance. In addition, PA resistance cosegregated in ‘Jester’ with a single dominant gene for BGA resistance. These results raised the possibility that both resistances may be mediated by the same mechanism. This was not supported by the results of gene induction studies, and resistance induced by BGA had no effect on PA feeding. Moreover, different genetic backgrounds containing a BGA resistance gene from the same resistance donor differ in resistance to PA. These results suggest that distinct mechanisms are involved in resistance to these two aphid species. Resistance to PA and BGA in the same genetic background in M. truncatula makes this plant an attractive model for the study of both plant and aphid components of resistant and susceptible plant-aphid interactions.

A single gene, AIN, in Medicago truncatula mediates a hypersensitive response to both bluegreen aphid and pea aphid, but confers resistance only to bluegreen aphid

Biotic stress in plants frequently induces a hypersensitive response (HR). This distinctive reaction has been studied intensively in several pathosystems and has shed light on the biology of defence signalling. Compared with microbial pathogens, relatively little is known about the role of the HR in defence against insects. Reference genotype A17 of Medicago truncatula Gaertn., a model legume, responds to aphids of the genus Acyrthosiphon with necrotic lesions resembling a HR. In this study, the biochemical nature of this response, its mode of inheritance, and its relationship with defence against aphids were investigated. The necrotic lesion phenotype and resistance to the bluegreen aphid (BGA, Acyrthosiphon kondoi Shinji) and the pea aphid (PA, Acyrthosiphon pisum (Harris)) were analysed using reference genotypes A17 and A20, their F2 progeny and recombinant inbred lines. BGA-induced necrotic lesions co-localized with the production of H2O2, consistent with an oxidative burst widely associated with hypersensitivity. This HR correlated with stronger resistance to BGA in A17 than in A20; these phenotypes cosegregated as a semi-dominant gene, AIN (Acyrthosiphon-induced necrosis). In contrast to BGA, stronger resistance to PA in A17, compared with A20, did not cosegregate with a PA-induced HR. The AIN locus resides in a cluster of sequences predicted to encode the CC-NBS-LRR subfamily of resistance proteins. AIN-mediated resistance presents a novel opportunity to use a model plant and model aphid to study the role of the HR in defence responses to phloem-feeding insects.

Control of cell proliferation, endoreduplication, cell size, and cell death by the retinoblastoma-related pathway in maize endosperm

Significance Cereal endosperm is a key source of dietary calories and raw materials for countless manufactured goods. Understanding how the cell cycle is regulated during endosperm development could lead to increased crop yield. We show that a maize Retinoblastoma-related gene, RBR1, plays a central role in regulating gene expression, endoreduplication, and the number, size, and death of endosperm cells. RBR1 is genetically coupled to Cyclin Dependent Kinase A;1 in controlling endoreduplication but not gene expression. Seeds down-regulated for RBR1 develop normally, which suggests higher-order control mechanisms regulating endosperm development that are superimposed on cell cycle regulation. The endosperm of cereal grains is one of the most valuable products of modern agriculture. Cereal endosperm development comprises different phases characterized by mitotic cell proliferation, endoreduplication, the accumulation of storage compounds, and programmed cell death. Although manipulation of these processes could maximize grain yield, how they are regulated and integrated is poorly understood. We show that the Retinoblastoma-related (RBR) pathway controls key aspects of endosperm development in maize. Down-regulation of RBR1 by RNAi resulted in up-regulation of RBR3-type genes, as well as the MINICHROMOSOME MAINTENANCE 2–7 gene family and PROLIFERATING CELL NUCLEAR ANTIGEN, which encode essential DNA replication factors. Both the mitotic and endoreduplication cell cycles were stimulated. Developing transgenic endosperm contained 42–58% more cells and ∼70% more DNA than wild type, whereas there was a reduction in cell and nuclear sizes. In addition, cell death was enhanced. The DNA content of mature endosperm increased 43% upon RBR1 down-regulation, whereas storage protein content and kernel weight were essentially not affected. Down-regulation of both RBR1 and CYCLIN DEPENDENT KINASE A (CDKA);1 indicated that CDKA;1 is epistatic to RBR1 and controls endoreduplication through an RBR1-dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback loop. These results indicate that the RBR1 pathway plays a major role in regulation of different processes during maize endosperm development and suggest the presence of tissue/organ-level regulation of endosperm/seed homeostasis.

Identification of distinct quantitative trait loci associated with defence against the closely related aphids Acyrthosiphon pisum and A. kondoi in Medicago truncatula

Aphids are a major family of plant insect pests. Medicago truncatula and Acyrthosiphon pisum (pea aphid, PA) are model species with a suite of resources available to help dissect the mechanism underlying plant–aphid interactions. A previous study focused on monogenic and relatively strong resistance in M. truncatula to PA and other aphid species. In this study a moderate resistance to PA was characterized in detail in the M. truncatula line A17 and compared with the highly susceptible line A20 and the more resistant line Jester. The results show that PA resistance in A17 involves both antibiosis and tolerance, and that resistance is phloem based. Quantitative trait locus (QTL) analysis using a recombinant inbred line (RIL) population (n=114) from a cross between A17 and A20 revealed that one locus, which co-segregated with AIN (Acyrthosiphon-induced necrosis) on chromosome 3, is responsible for the reduction of aphid biomass (indicator of antibiosis) for both PA and bluegreen aphid (BGA, A. kondoi), albeit to a lesser degree for PA than BGA. Interestingly, two independent loci on chromosomes 5 and 3 were identified for the plant biomass reduction (indicator of plant tolerance) by PA and BGA, respectively, demonstrating that the plant’s tolerance response to these two closely related aphid species is distinct. Together with previously identified major resistant (R) genes, the QTLs identified in this study are powerful tools to understand fully the spectrum of plant defence against sap-sucking insects and provide opportunities for breeders to generate effective and sustainable strategies for aphid control.

Characterization and genetic dissection of resistance to spotted alfalfa aphid (Therioaphis trifolii) in Medicago truncatula.

Aphids cause significant yield losses in agricultural crops worldwide. Medicago truncatula, a model legume, cultivated pasture species in Australia and close relative of alfalfa (Medicago sativa), was used to study the defence response against Therioaphis trifolii f. maculate [spotted alfalfa aphid (SAA)]. Aphid performance and plant damage were compared among three accessions. A20 is highly susceptible, A17 has moderate resistance, and Jester is strongly resistant. Subsequent analyses using A17 and A20, reciprocal F1s and an A17×A20 recombinant inbred line (RIL) population revealed that this moderate resistance is phloem mediated and involves antibiosis and tolerance but not antixenosis. Electrical penetration graph analysis also identified a novel waveform termed extended potential drop, which occurred following SAA infestation of M. truncatula. Genetic dissection using the RIL population revealed three quantitative trait loci on chromosomes 3, 6, and 7 involved in distinct modes of aphid defence including antibiosis and tolerance. An antibiosis locus resides on linkage group 3 (LG3) and is derived from A17, whereas a plant tolerance and antibiosis locus resides on LG6 and is derived from A20, which exhibits strong temporary tolerance. The loci identified reside in regions harbouring classical resistance genes, and introgression of these loci in current medic cultivars may help provide durable resistance to SAA, while elucidation of their molecular mechanisms may provide valuable insight into other aphid–plant interactions.

Identification of resistance to bluegreen aphid and spotted alfalfa aphid in Trigonella species

Abstract The genus Trigonella (Fabaceae, tribe Trifolieae) includes potential new species for pasture legume production in phase farming systems of southern Australia. Performance under biotic stress is an important factor in selecting the most suitable species and genotypes for pasture legume breeding programs. Fifty‐nine accessions representing 26 Trigonella species and two Medicago species (formerly with Trigonella) were evaluated for resistance to two aphid species, bluegreen aphid (Acyrthosiphon kondoi Shinji), and spotted alfalfa aphid (Therioaphis trifolii (Monell) fm. maculata) in glasshouse experiments. An accession of Trigonella macrorrhyncha (SA 32223) was found to be highly resistant to both the aphids. Accessions of T. coerulescens, T. cylindracea, T. gladiata, T. mesopotamica, T. schlumbergeri and Medicago monspeliaca were found to be resistant to bluegreen aphid, but susceptible to spotted alfalfa aphid. The resistance mechanism in T. macrorrhyncha involved both antixenosis (non‐preference in a choice test) and antibiosis (reduced fecundity). The implications of these results for germplasm improvement are discussed.