Application of biotechnology in breeding lentil for resistance to biotic and abiotic stress

Lentil is a self-pollinating diploid (2n = 14 chromosomes) annual cool season legume crop that is produced throughout the world and is highly valued as a high protein food. Several abiotic stresses are important to lentil yields world wide and include drought, heat, salt susceptibility and iron deficiency. The biotic stresses are numerous and include: susceptibility to Ascochyta blight, caused by Ascochyta lentis; Anthracnose, caused by Colletotrichum truncatum; Fusarium wilt, caused by Fusarium oxysporum; Sclerotinia white mold, caused by Sclerotinia sclerotiorum; rust, caused by Uromyces fabae; and numerous aphid transmitted viruses. Lentil is also highly susceptible to several species of Orabanche prevalent in the Mediterranean region, for which there does not appear to be much resistance in the germplasm. Plant breeders and geneticists have addressed these stresses by identifying resistant/tolerant germplasm, determining the genetics involved and the genetic map positions of the resistant genes. To this end progress has been made in mapping the lentil genome and several genetic maps are available that eventually will lead to the development of a consensus map for lentil. Marker density has been limited in the published genetic maps and there is a distinct lack of co-dominant markers that would facilitate comparisons of the available genetic maps and efficient identification of markers closely linked to genes of interest. Molecular breeding of lentil for disease resistance genes using marker assisted selection, particularly for resistance to Ascochyta blight and Anthracnose, is underway in Australia and Canada and promising results have been obtained. Comparative genomics and synteny analyses with closely related legumes promises to further advance the knowledge of the lentil genome and provide lentil breeders with additional genes and selectable markers for use in marker assisted selection. Genomic tools such as macro and micro arrays, reverse genetics and genetic transformation are emerging technologies that may eventually be available for use in lentil crop improvement.

Transfer of an autonomously replicating vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides

Previous research has indicated that biotypes A and B of Colletotrichum gloeosporioides that infect Stylosanthes spp. in Australia are asexual and vegetatively incompatible. Selectable marker genes conferring resistance either to hygromycin or phleomycin were introduced into isolates of these biotypes. Vectors conferring resistance to hygromycin and carrying telomeric sequences from Fusarium oxysporum replicated autonomously in C. gloeosporioides and gave frequencies of transformation 100-times higher than vectors that integrated into the genome. Monoconidial colonies resistant to both antibiotics were recovered when hygromycin-resistant biotype-A transformants carrying an autonomously replicating vector were paired in culture with a phleomycin-resistant biotype-B transformant carrying integrative vector sequences. Molecular analysis of double antibiotic-resistant progeny indicated that they contained the autonomous vector in a biotype-B genetic background, Results indicate that transfer of the autonomous vector had occurred from biotype A to biotype B, demonstrating the potential for transfer of genetic information between these biotypes.

Saprophytic microorganisms with potential for biological control of Botrytis cinerea on Geraldton waxflower flowers

Saprophytic bacteria, yeasts and filamentous fungi were isolated from Geraldton waxflower flowers and screened to identify potential antagonism towards Botrytis cinerea. Isolates from other sources (e.g. avocado) were also tested. Isolates were initially screened in vitro for inhibition of B. cinerea conidial germination, germ tube elongation and mycelial growth. The most antagonistic bacteria, yeasts and fungi were selected for further testing on detached waxflower flowers. Conidia of the pathogen were mixed with conidia or cells of the selected antagonists, co-inoculated onto waxflower flowers, and the flowers were sealed in glass jars and incubated at 20 degreesC. The number of days required for the pathogen to cause flower abscission was determined. The most antagonistic bacterial isolate, Pseudomonas sp. 677, significantly reduced conidial germination and retarded germ tube elongation of B. cinerea. None of the yeast or fungal isolates tested was found to significantly reduce conidial germination or retard germ tube elongation, but several significantly inhibited growth of B. cinerea. Fusarium sp., Epicoccum sp. and Trichoderma spp. were the most antagonistic of these isolates. Of the isolates tested on waxflower, Pseudomonas sp. 677 was highly antagonistic towards B. cinerea and delayed waxflower abscission by about 3 days. Trichoderma harzianum also significantly delayed flower abscission. However, as with most of the fungal antagonists used, inoculation of waxflower flowers with this isolate resulted in unsightly mycelial growth.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Heterotrimeric G proteins facilitate Arabidopsis resistance to necrotrophic pathogens and are involved in jasmonate signaling

Heterotrimeric G proteinshave been previously linked to plant defense; however a role for the G beta gamma dimer in defense signaling has not been described to date. Using available Arabidopsis (Arabidopsis thaliana) mutants lacking functional G alpha or G beta subunits, we show that defense against the necrotrophic pathogens Alternaria brassicicola and Fusarium oxysporum is impaired in G beta- deficient mutants while G alpha-deficient mutants show slightly increased resistance compared to wild-type Columbia ecotype plants. In contrast, responses to virulent (DC3000) and avirulent (JL1065) strains of Pseudomonas syringae appear to be independent of heterotrimeric G proteins. The induction of a number of defense-related genes in G beta-deficient mutants were severely reduced in response to A. brassicicola infection. In addition, G beta-deficient mutants exhibit decreased sensitivity to a number of methyl jasmonate- induced responses such as induction of the plant defensin gene PDF1.2, inhibition of root elongation, seed germination, and growth of plants in sublethal concentrations of methyl jasmonate. In all cases, the behavior of the G alpha- deficient mutants is coherent with the classic heterotrimeric mechanism of action, indicating that jasmonic acid signaling is influenced by the Gbg functional subunit but not by G alpha. We hypothesize that G beta gamma acts as a direct or indirect enhancer of the jasmonate signaling pathway in plants.