Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate

We investigated what gene(s) in the plant roots have the positive role against repressing root-knot nematode (RKN) infection. We investigated the interaction between RKN infection and gene expression in the plant roots induced by methyl jasmonate (MeJA). We focused on the induced resistance response and the duration after foliar treatment with MeJA of 0.1, 0.5, 1.0, and 5.0mM at 1, 24, 48, and 72h prior to the inoculation of RKN. As a result, the foliar treatment with MeJA at 0.5mM or higher concentrations significantly reduced the infection of RKN in plants and the effect lasted for about 1 week. The repressing effect on RKN population declined to the lowest level in two weeks after MeJA treatment. The expression of proteinase inhibitors (PIs) and multicystatin (MC) were induced while the repressing effect on RKN was valid and a negative correlation was found between the expression of PIs or MC and RKN infection. In addition, when tomato plants no longer expressing MC and PIs were treated again with MeJA, the repressing effect revived. These phenomena appeared to be regardless of the existence of Mi-genes or isolate of RKN. Our results indicate that the expression level of MC and PIs may be effective as marker genes for estimating the induced resistance response against RKN infection.     

Post-transcriptional gene silencing of root-knot nematode in transformed soybean roots

RNAi constructs targeted to four different genes were examined to determine their efficacy to reduce galls formed by Meloidogyne incognita in soybean roots. These genes have high similarity with essential soybean cyst nematode (Heterodera glycines) and Caenorhabditis elegans genes. Transformed roots were challenged with M. incognita. Two constructs, targeted to genes encoding tyrosine phosphatase (TP) and mitochondrial stress-70 protein precursor (MSP), respectively, strongly interfered with M. incognita gall formation. The number of galls formed on roots transformed with constructs targeting the M. incognita TP and MSP genes was reduced by 92% and 94.7%, respectively. The diameter of M. incognita inside these transformed roots was 5.4 and 6.5 times less than the diameter of M. incognita found inside control plants transformed with the empty vector. These results indicate that silencing the genes encoding TP and MSP can greatly decrease gall formation and shows a promising solution for broadening resistance of plants against this plant-parasitic nematode.     

Phosphonate fertilizers suppressed root knot nematodes Meloidogyne javanica and M. incognita

The efficacy of the phosphonate fertilizers, Calphos^® (a.i. calcium phosphonate), Magphos^® (a.i. magnesium phosphonate and potassium phosphonate) and Phosphoros^® (a.i. potassium phosphonate) against two species of root knot nematodes (RKN), Meloidogyne javanica and M. incognita is evaluated. Laboratory experiments showed that Calphos^®, Magphos^® and their main components inhibited egg hatching and caused 100% mortality of the second stage juveniles (J2s) of the two RKN species; the hatching inhibition effects persisted after transferring the egg masses of both species to water. However, Phosphoros^® (0.5%) did not suppress egg hatching or the survival of J2s of both RKN species. No hatching occurred when egg masses were treated for one week with the nematicide Vydate L^® (2 ml/l), however, J2s hatched when the Vydate L^® treated egg masses were moved to water. The glasshouse study indicated that Magphos^®, Calphos^® and Phosphoros^® reduced root galling caused by M. javanica by 98, 66 and 47%, respectively, in comparison to the untreated controls. Magphos^® resulted in the lowest number of root galls formed by M. incognita, the reduction was 84%. In contrast, Calphos^® and Phosphoros^® reduced galling by 47 and 39%, respectively. The Magphos^® treatment resulted in the lowest numbers of egg masses and the lowest reproductive factor (RF) of both nematode species. However, plants treated with Phosphoros^® resulted in higher foliage weights compared with the application of the other two fertilizers and the untreated plants.     

RKN Lethal DB: A database for the identification of Root Knot Nematode (Meloidogyne spp.) candidate lethal genes

Root Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating parasites that infect the roots of hundreds of plant species. RKN cannot live independently from their hosts and are the biggest contributors to the loss of the world''s primary foods. RNAi gene silencing studies have demonstrated that there are fewer galls and galls are smaller when RNAi constructs targeted to silence certain RKN genes are expressed in plant roots. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne Arenaria, Meloidogyne Chitwoodi, Meloidogyne Hapla, Meloidogyne Incognita, Meloidogyne Javanica, and Meloidogyne Paranaensis to that of the free living nematode Caenorhabditis elegans, to identify candidate genes that will be lethal to RKN when silenced or mutated. Our analysis yielded a number of such candidate lethal genes in RKN, some of which have been tested and proven to be effective in soybean roots. A web based database was built to house and allow scientists to search the data. This database will be useful to scientists seeking to identify candidate genes as targets for gene silencing to confer resistance in plants to RKN.

Availability

The database can be accessed from http://bioinformatics.towson.edu/RKN/