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.     

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.     

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.     

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.     

利用脂肪酸生物标记对尖镰孢寄主专化型的快速鉴定

尖镰孢寄主范围广、遗传差异大,其种下存在多种寄主专化型。对尖镰孢寄主专化型的快速鉴定可为科学制定植物病害防控策略提供依据。利用Sherlock MIS脂肪酸鉴定系统对分离自番茄、棉花、黄瓜、茄子等4种寄主专化型的18株尖镰孢进行脂肪酸成分测定,共检测到10种脂肪酸。运用SPSS软件中的PCA法对被检测到的脂肪酸进行主成分分析,确定了18:1CIS9（W9）[X1],18:2 CIS 9,12/18:0a[X2]和18:00[X3]等3个脂肪酸为其主成分。利用Bayes逐步判别法建立了尖镰孢4种不同寄主专化型判别模型为Y1=-157.750+2.809X1+3.391X2+8.099X3;Y2=-178.343+0.586X1+7.587X2- 0.214X3;Y3=-129.132+2.749X1+4.163X2+4.476X3;Y4=-201.307+2.016X1+7.345X2+2.400X3。通过对43株未知寄主专化型菌株主成分脂肪酸的测定,利用判别法对尖镰孢进行判定,结果发现有40株与原寄主来源一致,判对率达93%。表明脂肪酸生物标记法可用于尖镰孢寄主专化型的快速鉴定。     

Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells

Fusarium oxysporum is an asexual, soil inhabiting fungus that comprises many different formae speciales, each pathogenic towards a different host plant. In absence of a suitable host all F. oxysporum isolates appear to have a very similar lifestyle, feeding on plant debris and colonizing the rhizosphere of living plants. Upon infection F. oxysporum switches from a saprophytic to an infectious lifestyle, which probably includes the reprogramming of gene expression. In this work we show that the expression of the known effector gene SIX1 of F. oxysporum f. sp. lycopersici is strongly upregulated during colonization of the host plant. Using GFP (green fluorescent protein) as reporter, we show that induction of SIX1 expression starts immediately upon penetration of the root cortex. Induction requires living plant cells, but is not host specific and does not depend on morphological features of roots, since plant cells in culture can also induce SIX1 expression. Taken together, F. oxysporum seems to be able to distinguish between living and dead plant material, preventing unnecessary switches from a saprophytic to an infectious lifestyle.     

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.     

A small, cysteine-rich protein secreted by Fusarium oxysporum during colonization of xylem vessels is required for I-3-mediated resistance in tomato

A 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wild-type strain results in breaking of I-3-mediated resistance. These results suggest that I-3-mediated resistance is based on recognition of Six1 secreted in xylem vessels.     

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.     

Identification of F. o. cucumerinum and F. o. luffae by RAPD-generated DNA probes

AIMS: To create a fast, sensitive and specific method for identifying Fusarium oxysporum f. sp. cucumerinum and F. o. luffae. METHODS AND RESULTS: Specific DNA bands were selected as probes from RAPD profiles of 13 formae speciales of F. oxysporum. The forma specialis-specific probe OPC18300c and OPC18520f could be used to identify F. o. cucumerinum and F. o. luffae by RAPD-PCR followed dot blot hybridization, respectively. CONCLUSIONS: A specific method for identifying F. o. cucumerinum and F. o. luffae was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: F. oxysporum formae speciales identification with a DNA probe can be relatively rapid and provides a method to identify the pathogen without host inoculation tests.     

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.     

The effector protein Avr2 of the xylem-colonizing fungus Fusarium oxysporum activates the tomato resistance protein I-2 intracellularly

To promote host colonization, many plant pathogens secrete effector proteins that either suppress or counteract host defences. However, when these effectors are recognized by the host's innate immune system, they trigger resistance rather than promoting virulence. Effectors are therefore key molecules in determining disease susceptibility or resistance. We show here that Avr2, secreted by the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici ( Fol ), shows both activities: it is required for full virulence in a susceptible host and also triggers resistance in tomato plants carrying the resistance gene I-2 . Point mutations in AVR2 , causing single amino acid changes, are associated with I-2 -breaking Fol strains. These point mutations prevent recognition by I-2 , both in tomato and when both genes are co-expressed in leaves of Nicotiana benthamiana . Fol strains carrying the Avr2 variants are equally virulent, showing that virulence and avirulence functions can be uncoupled. Although Avr2 is secreted into the xylem sap when Fol colonizes tomato, the Avr2 protein can be recognized intracellularly by I-2, implying uptake by host cells.     

Pantothenate synthetase from Fusarium oxysporum f. sp. lycopersici is induced by alpha-tomatine

The steroidal glycoalkaloid alpha-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and purified to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, beta-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by alpha-tomatine.     

Characterization of the formae speciales of Fusarium oxysporum causing wilts of cucurbits by DNA fingerprinting with nuclear repetitive DNA sequences.

The genetic relatedness of five formae speciales of Fusarium oxysporum causing wilts of cucurbit plants was determined by DNA fingerprinting with the moderately repetitive DNA sequences FOLR1 to FOLR4. The four FOLR clones were chosen from a genomic library made from F. oxysporum f. sp. lagenariae 03-05118. Total DNAs from 50 strains representing five cucurbit-infecting formae speciales, cucumerinum, melonis, lagenariae, niveum, and momordicae, and 6 strains of formae speciales pathogenic to other plants were digested with EcoRV and hybridized with 32P-labeled FOLR probes. The strains were clearly distinguishable at the formae specialis level on the basis of FOLR DNA fingerprints. Fifty-two fingerprint types were detected among the 56 strains by using all FOLR probes. These probes were used to infer phylogenetic relationships among the DNA fingerprint types by the unweighted pair group method using averages and parsimony analysis. The fingerprint types detected in each of the formae speciales cucumerinum, lagenariae, niveum, and momordicae were grouped into a single cluster. However, two different genetic groups occurred in the formae specialis melonis. The two groups also differed in pathogenicity: one group caused wilts of muskmelon and oriental melon, while the second was pathogenic only to muskmelon. The fingerprint types of different formae speciales pathogenic to plants other than cucurbits were distinguishable from one another and from the fingerprints of the cucurbit-infecting strains. These results suggest that the cucurbit-infecting formae speciales are intraspecific variants distinguishable at the DNA level and in their host range.     

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.