Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-alpha1,4-polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.     

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.     

Tomatinase from Fusarium oxysporum f. sp. lycopersici defines a new class of saponinases.

Plants produce a variety of secondary metabolites, many of which have antifungal activity. Saponins are plant glycosides that may provide a preformed chemical barrier against phytopathogenic fungi. Fusarium oxysporum f. sp. lycopersici and other tomato pathogens produce extracellular enzymes known as tomatinases, which deglycosylate alpha-tomatine to yield less toxic derivatives. We have cloned and characterized the cDNA and genomic DNA encoding tomatinase from the vascular pathogen of tomato F. oxysporum f. sp. lycopersici. This gene encodes a protein (FoTom1) with no amino acid sequence homology to any previously described saponinase, including tomatinase from Septoria lycopersici. Although FoTom1 is related to family 10 glycosyl hydrolases, which include mainly xylanases, it has no detectable xylanase activity. We have overexpressed and purified the protein with a bacterial heterologous system. The purified enzyme is active and cleaves alpha-tomatine into the less toxic compounds tomatidine and lycotetraose. Tomatinase from F. oxysporum f. sp. lycopersici is encoded by a single gene whose expression is induced by alpha-tomatine. This expression is fully repressed in the presence of glucose, which is consistent with the presence of two putative CREA binding sites in the promoter region of the tomatinase gene. The tomatinase gene is expressed in planta in both roots and stems throughout the entire disease cycle of F. oxysporum f. sp. lycopersici.     

A highly conserved effector in Fusarium oxysporum is required for full virulence on Arabidopsis

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Δsix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Δsix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.     

Tomatinase from Fusarium oxysporum f. sp. lycopersici is required for full virulence on tomato plants

Saponin detoxification enzymes from pathogenic fungi are involved in the infection process of their host plants. Fusarium oxysporum f. sp lycopersici, a tomato pathogen, produces the tomatinase enzyme Tom1, which degrades alpha-tomatine to less toxic derivates. To study the role of the tom1 gene in the virulence of F. oxysporum, we performed targeted disruption and overexpression of the gene. The infection process of tomato plants inoculated with transformants constitutively producing Tom1 resulted in an increase of symptom development. By contrast, tomato plants infected with the knockout mutants showed a delay in the disease process, indicating that Tom1, although not essential for pathogenicity, is required for the full virulence of F. oxysporum. Total tomatinase activity in the disrupted strains was reduced only 25%, leading to beta(2)-tomatine as the main hydrolysis product of the saponin in vitro. In silico analysis of the F. oxysporum genome revealed the existence of four additional putative tomatinase genes with identities to tomatinases from family 3 of glycosyl hydrolases. These might be responsible for the remaining tomatinase activity in the Deltatom1 mutants. Our results indicate that detoxification of alpha-tomatine in F. oxysporum is carried out by several tomatinase activities, suggesting the importance of these enzymes during the infection process.     

Infectivity of Chlamydospores vs Microconidia of Fusarium oxysporum f.sp. lycopersici on Tomato

The effect of nature of inoculum on disease induced by Fusarium oxysporum f.sp. lycopersici on tomato was tested. Chlamydospores produced in soil 30 days after inoculation induced a more severe disease than microconidia indicating a higher inoculum potential of chlamydospores.
The method proposed produces easily an inoculum of F. oxysporum f.sp. lycopersici which infects the plants consistently and induces a relatively high disease severity.     

Plant growth-promoting rhizobacteria, Paenibacillus polymyxa and Paenibacillus lentimorbus suppress disease complex caused by root-knot nematode and fusarium wilt fungus

Aim:  To screen and evaluate the biocontrol potential of Paenibacillus strains against disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici interactions.
Methods and Results:  Paenibacillus strains were collected from rotten ginseng roots. The strains were tested under in vitro and pots for their inhibitory activities, and biocontrol potential against disease complex caused by M. incognita and F. oxysporum f. sp. lycopersici on tomato. In in vitro experiments, among 40 tested strains of Paenibacillus spp., 11 strains showed antifungal and nematicidal activities against F. oxysporum f. sp. lycopersici and M. incognita, respectively. Paenibacillus polymyxa GBR-462; GBR-508 and P. lentimorbus GBR-158 showed the strongest antifungal and nematicidal activities. These three strains used in pot experiment reduced the symptom development of the disease complex (wilting and plant death), and increased plant growth. The control effects were estimated to be 90–98%, and also reduced root gall formation by 64–88% compared to the untreated control.
Conclusion:  The protective properties of selected Paenibacillus strains make them as potential tool to reduce deleterious impact of disease complex plants.
Significance and Impact of the Study:  The study highlights biocontrol potential of Paenibacillus strains in management of disease complex caused by nematode-fungus interaction.     

Plant colonization by the vascular wilt fungus Fusarium oxysporum requires FOW1, a gene encoding a mitochondrial protein

The soil-borne fungus Fusarium oxysporum causes vascular wilts of a wide variety of plant species by directly penetrating roots and colonizing the vascular tissue. The pathogenicity mutant B60 of the melon wilt pathogen F. oxysporum f. sp. melonis was isolated previously by restriction enzyme-mediated DNA integration mutagenesis. Molecular analysis of B60 identified the affected gene, designated FOW1, which encodes a protein with strong similarity to mitochondrial carrier proteins of yeast. Although the FOW1 insertional mutant and gene-targeted mutants showed normal growth and conidiation in culture, they showed markedly reduced virulence as a result of a defect in the ability to colonize the plant tissue. Mitochondrial import of Fow1 was verified using strains expressing the Fow1-green fluorescent protein fusion proteins. The FOW1-targeted mutants of the tomato wilt pathogen F. oxysporum f. sp. lycopersici also showed reduced virulence. These data strongly suggest that FOW1 encodes a mitochondrial carrier protein that is required specifically for colonization in the plant tissue by F. oxysporum.     

The arms race between tomato and Fusarium oxysporum

The interaction between tomato and Fusarium oxysporum f. sp. lycopersici has become a model system for the study of the molecular basis of disease resistance and susceptibility. Gene-for-gene interactions in this system have provided the basis for the development of tomato cultivars resistant to Fusarium wilt disease. Over the last 6 years, new insights into the molecular basis of these gene-for-gene interactions have been obtained. Highlights are the identification of three avirulence genes in F. oxysporum f. sp. lycopersici and the development of a molecular switch model for I-2, a nucleotide-binding and leucine-rich repeat-type resistance protein which mediates the recognition of the Avr2 protein. We summarize these findings here and present possible scenarios for the ongoing molecular arms race between tomato and F. oxysporum f. sp. lycopersici in both nature and agriculture.     

Fusarium oxysporum evades I-3-mediated resistance without altering the matching avirulence gene

I-3-Mediated resistance of tomato against Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici depends on Six1, a protein that is secreted by the fungus during colonization of the xylem. Among natural isolates of F. oxysporum f. sp. lycopersici are several that are virulent on a tomato line carrying only the I-3 resistance gene. However, evasion of I-3-mediated resistance by these isolates is not correlated with mutation of the SIX1 gene. Moreover, the SIX1 gene of an I-3-virulent isolate was shown to be fully functional in that i) the gene product is secreted in xylem sap, ii) deletion leads to a further increase in virulence on the I-3 line as well as reduced virulence on susceptible lines, and iii) the gene confers full avirulence on the I-3 line when transferred to another genetic background. Remarkably, all I-3-virulent isolates were of race 1, suggesting a link between the presence of AVR1 and evasion of I-3-mediated resistance.     

A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum

The fungal species Fusarium oxysporum is a ubiquitous inhabitant of soils worldwide that includes pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. However, the genetic basis of host specificity and virulence in F. oxysporum is so far unknown. In this study, a robust random-amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. While the F. oxysporum radicis-cucumerinum strains were found to cluster in a separate clade based on elongation factor-1alpha phylogeny, strains belonging to F. oxysporum f. sp. cucumerinum were found to be genetically more diverse. This is reflected in the observation that specificity testing of the identified markers using a broad collection of F. oxysporum strains with all known vegetative compatibility groups of the target formae speciales, as well as representative strains belonging to other formae speciales, resulted in two cross-reactions for the F. oxysporum f. sp. cucumerimum marker. However, no cross-reactions were observed for the F. oxysporum f. sp. radicis-cucumerimum marker. This F. oxysporum f. sp. radicis-cucumerimum marker shows homology to Folyt1, a transposable element identified in the tomato pathogen F. oxysporum f. sp. lycopersici and may possibly play a role in host-range specificity in the target forma specialis. The markers were implemented in a DNA array that enabled parallel and sensitive detection and identification of the pathogens in complex samples from diverse origins.     

Interaction between Meloidogyne incognita and Agrobacterium tumefaciens or Fusarium oxysporum f. sp. lycopersici on Tomato

Agrobacterium tumefaciens stimulated and Fusarium oxysporum f. sp. lycopersici inhibited development and reproduction of Meloidogyne incognita when applied to the opposite split root of tomato, Lycopersicon esculentum cv. Tropic, plants. The lowest rate of nematode reproduction occurred after 2,000 juveniles were applied and the fungus was present in the opposite split root. The effects of all three pathogens alone on the growth of roots and shoots of tomato plants were evident, but M. incognita had a greater effect alone than did either of the other pathogens. The length of split roots was reduced by the infection of M. incognita and A. tumefaciens or F. oxysporum f. sp. lycopersici. The number of galls induced by nematodes on roots was higher where the bacterium was applied and lower where the fungus was applied to the opposite split root.     

The I2C family from the wilt disease resistance locus I2 belongs to the nucleotide binding, leucine-rich repeat superfamily of plant resistance genes.

Characterization of plant resistance genes is an important step in understanding plant defense mechanisms. Fusarium oxysporum f sp lycopersici is the causal agent of a vascular wilt disease in tomato. Genes conferring resistance to plant vascular diseases have yet to be described molecularly. Members of a new multigene family, complex I2C, were isolated by map-based cloning from the I2 F. o. lycopersici race 2 resistance locus. The genes show structural similarity to the group of recently isolated resistance genes that contain a nucleotide binding motif and leucine-rich repeats. Importantly, the presence of I2C antisense transgenes abrogated race 2 but not race 1 resistance in otherwise normal plants. Expression of the complete sense I2C-1 transgene conferred significant but partial resistance to F. o. lycopersici race 2. All members of the I2C gene family have been mapped genetically and are dispersed on three different chromosomes. Some of the I2C members cosegregate with other tomato resistance loci. Comparison within the leucine-rich repeat region of I2C gene family members shows that they differ from each other mainly by insertions or deletions.     

Effector gene screening allows unambiguous identification of Fusarium oxysporum f. sp. lycopersici races and discrimination from other formae speciales

During infection of tomato, the fungus Fusarium oxysporum f. sp. lycopersici secretes several unique proteins, called 'secreted in xylem' (Six) proteins, into the xylem sap. At least some of these proteins promote virulence towards tomato and among them, all predicted avirulence proteins that can trigger disease resistance in tomato have been found. In this study, a large, worldwide collection of F. oxysporum isolates was screened for the presence of seven SIX genes ( SIX1 – SIX7 ). The results convincingly show that identification of F. oxysporum formae speciales and races based on host-specific virulence genes can be very robust. SIX1, SIX2, SIX3 and SIX5 can be used for unambiguous identification of the forma specialis lycopersici . In addition, SIX4 can be used for the identification of race 1 strains, while polymorphisms in SIX3 can be exploited to differentiate race 2 from race 3 strains. For SIX6 and SIX7 , close homologs were found in a few other formae speciales , suggesting that these genes may play a more general role in pathogenicity. Host specificity may be determined by the unique SIX genes, possibly in combination with the absence of genes that trigger resistance in the host.     

Genome-wide dissection of<Emphasis Type="Italic"> Fusarium</Emphasis> resistance in tomato reveals multiple complex loci

Resistance to different pathogenic races of Fusarium oxysporum f. sp. lycopersici (F. o. lycopersici) was explored at two genomic levels in tomato. Six independent Fusarium resistance loci were identified by comparing the responses of a complete set of 53 lines carrying different introgressed regions of the Lycopersicon pennellii genome in a L. esculentum background. The loci confer varying degrees of resistance to different races of the pathogen. Corresponding map positions from different tomato species were aligned and in some cases revealed parallel resistance to F. o. lycopersici with qualitative changes in race specificities. One of the loci identified corresponds to the previously characterized complex resistance locus I2, which is involved in resistance to F. o. lycopersici race 2. A novel member of this locus, I2C-5, which belongs to the NBS-LRR family of resistance genes, was cloned and shown to confer partial resistance in transgenic plants. Thus, at a particular complex locus gene members can confer full or partial resistance to F. o. lycopersici race 2. The results of our whole-genome mapping analysis underline the robust independent origin of resistance to a particular disease and demonstrate the conservation of resistance features at syntenic loci, together with the rapid diversification of genes for innate resistance within loci.