Expression Analysis of Defense-Related Genes in Cotton (Gossypium hirsutum) after Fusarium oxysporum f. sp. vasinfectum Infection and Following Chemical Elicitation using a Salicylic Acid Analog and Methyl Jasmonate

Cotton (Gossypium hirsutum) wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov) is considered as a major threat for commercial cotton production worldwide. Relative expression ratios of two key pathogenesis-related (PR) genes (PR-3 and PR-10) and a detoxification gene (GST18) were compared between a fully susceptible (“LACTA”) and a partially field-resistant (“EMERALD”) cultivar after challenging with an Australian Fov isolate, as well as after pre-treatments with chemical inducers of defense such as BION® (a chemical analog of salicylic acid) and methyl-jasmonate (MeJA) prior to Fov inoculation. It was demonstrated that in both hypocotyls and roots of “EMERALD”, all PR genes were over-expressed after inoculation with Fov but not in the fully susceptible cultivar. Fov inoculation did not significantly affect GST18 expression in both cultivars. Exogenous application of each defense elicitor, prior to Fov inoculation, resulted in up-regulation of the three genes in root tissues of the fully susceptible cultivar. BION® application did not influence PR-3 expression in hypocotyls of both cultivars, whereas MeJA application resulted in induction of PR-3 in both cultivars. Furthermore, in hypocotyls of “LACTA”, over-expression of PR-10 was recorded after treatment with each chemical inducer. This pathogen exhibited different ability in eliciting oxidative burst in roots of the two cotton cultivars used in our analysis.     

The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov. Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants

Resistance conferred by the L³ gene is active against most ofthe tobamoviruses, including the Spanish strain (PMMoV-S), aP_1,2 pathotype, but not against certain strains of pepper mildmottle virus (PMMoV), termed P_1,2,3 pathotype, such as the Italianstrain (PMMoV-I). Both viruses are nearly identical at theirnucleotide sequence level (98%) and were used to challenge Capsicumchinense PI159236 plants harbouring the L³ gene in order tocarry out a comparative proteomic analysis of PR proteins inducedin this host in response to infection by either PMMoV-S or PMMoV-I.PMMoV-S induces a hypersensitive reaction (HR) in C. chinensePI159236 plant leaves with the formation of necrotic local lesionsand restriction of the virus at the primary infection sites.In this paper, C. chinense PR protein isoforms belonging tothe PR-1, β-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-likeprotein (PR-5), peroxidases (PR-9), germin-like protein (PR-16),and PRp27 (PR-17) have been identified. Three of these PR proteinisoforms were specifically induced during PMMoV-S-activationof C. chinense L³ gene-mediated resistance: an acidic β-1,3-glucanaseisoform (PR-2) (M_r 44.6; pI 5.1), an osmotin-like protein (PR-5)(M_r 26.8; pI 7.5), and a basic PR-1 protein isoform (M_r 18;pI 9.4–10.0). In addition, evidence is presented for adifferential accumulation of C. chinense PR proteins and mRNAsin the compatible (PMMoV-I)–C. chinense and incompatible(PMMoV-S)–C. chinense interactions for proteins belongingto all PR proteins detected. Except for an acidic chitinase(PR-3) (M_r 30.2; pI 5.0), an earlier and higher accumulationof PR proteins and mRNAs was detected in plants associated withHR induction. Furthermore, the accumulation rates of PR proteinsand mRNA did not correlate with maximal accumulation levelsof viral RNA, thus indicating that PR protein expression mayreflect the physiological status of the plant. Key words: Capsicum chinense, compatible interaction, incompatible interaction, HR-induction, PMMoV, PR proteins Received 5 December 2007; Revised 21 January 2008 Accepted 22 January 2008     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

Molecular mapping of a new source of Fusarium wilt resistance in tetraploid cotton (Gossypium hirsutum L.)

Fusarium wilt (FW) disease is an economically important disease of cotton worldwide and a major cause of crop losses in Australia and many other cotton-producing countries. Symptoms include wilting, vascular browning and death. Australian races of the causal agent Fusarium oxysporum f. sp. vasinfectum (Fov) are genetically distinct from those in other countries and are thought to have evolved from indigenous races. New sources of resistance for breeding are rare, as cotton cultivars with significant FW resistance against Fov isolates from other cotton-producing regions are usually susceptible to Australian Fov races. MCU-5, an Upland Indian cotton cultivar, has been identified as having improved resistance to Australian Fov and is being used to breed new commercial cultivars with higher resistance to FW. To investigate the genetic basis of the FW resistance in MCU-5, QTL analysis was performed on 244 F3 and 244 F4 families derived from an intraspecific cross between MCU-5 and Siokra 1-4, a cultivar highly sensitive to Australian Fov races. Resistance, as measured by leaf symptoms, vascular browning and survival, showed low to moderate heritability between generations. MCU-5 resistance to FW was found to be complex with three quantitative trait loci (QTL) identified in the F3, and eight in the F4, that explained between 9 and 41% of the phenotypic variation. The QTL were located on four linkage groups including chromosomes A6 (Chr 6), D4 (Chr 22) and D6 (Chr 25), with two QTL located in similar regions to previously identified FW resistance from the Sea Island cultivar Pima 3-79. The QTL identified in this study represent the first targets for marker-assisted selection of FW resistance in Australia.     

Fungicidal control of Rhizoctonia solani in soil amended with organic manures

Amendments of nutrient-deficient soil with three organic manures and one non-edible oil-cake reduced the disease controlling potential of methoxyethyl mercury chloride (MEMC), quintozene and carbendazim used as seed treatments on cowpea and cotton against seedling rot caused by Rhizoctonia solani. Biogas sludge (BGS) and farm yard manure (FYM) nullified the activity of MEMC and quintozene and reduced markedly the efficacy of carbendazim. Humic acid extracted from BGS inactivated MEMC and carbendazim but had little effect on quintozene. Green manure (Sesbania aculeata) slightly reduced the efficacy of MEMC only. Soil amendment with mahua (Madhuca indica) cake and soil drench with its aqueous extract greatly reduced the efficacy of the three fungicides.     

The effect of <Emphasis Type="Italic">Gossypium</Emphasis> C-genome chromosomes on resistance to fusarium wilt in allotetraploid cotton

Fusarium oxysporum f. sp. vasinfectum (Fov) has the potential to become the most economically significant pathogen of cotton in Australia. Although the levels of resistance present in the new commercial cultivars have improved significantly, they are still not immune and cotton breeders continue to look for additional sources of resistance. The native Australian Gossypium species represent an alternative source of resistance because they could have co-evolved with the indigenous Fov pathogens. Forty-six BC₃ G. hirsutum × G. sturtianum multiple alien-chromosome-addition-line (MACAL) families were challenged with a field-derived Fov isolate (VCG-01111). The G. hirsutum parent of the hexaploid MACAL is highly susceptible to fusarium wilt; the G. sturtianum parent is strongly resistant. Twenty-two of the BC₃ families showed enhanced fusarium wilt resistance relative to the susceptible G. hirsutum parent. Logistic regression identified four G. sturtianum linkage groups with a significant effect on fusarium wilt resistance: two linkage groups were associated with improved fusarium wilt resistance, while two linkage groups were associated with increased fusarium wilt susceptibility.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.