María D. García-Pedrajas

Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses

Severe Verticillium wilt of cotton in southern Spain is associated with the spread of a highly virulent, defoliating (D) pathotype of Verticillium dahliae. Eleven of the D and 15 of a mildly virulent, nondefoliating (ND) pathotype were analyzed by random amplified polymorphic DNA (RAPD) using the polymerase chain reaction (PCR). Six of 21 primers tested generated pathotype-associated RAPD bands. Another 21 V. dahliae isolates were compared in blind trials both by RAPD-PCR using the six selected primers and pathogenicity tests on cotton cultivars. There was a 100% correlation between pathotype characterization by each method. Unweighted paired group method with arithmetic averages cluster analysis was used to divide the 47 V. dahliae isolates into two clusters that correlated with the D or ND pathotypes. There was more diversity among ND isolates than among D isolates, these latter isolates being almost identical. ND- and D-associated RAPD bands of 2.0 and 1.0 kb, respectively, were cloned, sequenced, and used to design specific primers for the D and ND pathotypes. These pathotype-associated RAPD bands were present only in the genome of the pathotype from which they were amplified, as shown by Southern hybridization. The specific primers amplified only one DNA band of the expected size, and in the correct pathotype, when used for PCR with high annealing temperature. These specific primers successfully characterized V. dahliae cotton isolates from China and California as to D or ND pathotypes, thus demonstrating the validity and wide applicability of the results.

Genetics of morphogenesis and pathogenic development of Ustilago maydis.

Ustilago maydis has emerged as an important model system for the study of fungi. Like many fungi, U. maydis undergoes remarkable morphological transitions throughout its life cycle. Fusion of compatible, budding, haploid cells leads to the production of a filamentous dikaryon that penetrates and colonizes the plant, culminating in the production of diploid teliospores within fungal-induced plant galls or tumors. These dramatic morphological transitions are controlled by components of various signaling pathways, including the pheromone-responsive MAP kinase and cAMP/PKA (cyclic AMP/protein kinase A) pathways, which coregulate the dimorphic switch and sexual development of U. maydis. These signaling pathways must somehow cooperate with the regulation of the cytoskeletal and cell cycle machinery. In this chapter, we provide an overview of these processes from pheromone perception and mating to gall production and sporulation in planta. Emphasis is placed on the genetic determinants of morphogenesis and pathogenic development of U. maydis and on the fungus-host interaction. Additionally, we review advances in the development of tools to study U. maydis, including the recently available genome sequence. We conclude with a brief assessment of current challenges and future directions for the genetic study of U. maydis.

Class imbalance methods for translation initiation site recognition in DNA sequences

Translation initiation site (TIS) recognition is one of the first steps in gene structure prediction, and one of the common components in any gene recognition system. Many methods have been described in the literature to identify TIS in transcribed sequences such as mRNA, EST and cDNA sequences. However, the recognition of TIS in DNA sequences is a far more challenging task, and the methods described so far for transcripts achieve poor results in DNA sequences. Most methods approach this problem taking into account its biological characteristics. In this work we try a different view, considering this classification problem from a purely machine learning perspective. From the point of view of machine learning, TIS recognition is a class imbalance problem. Thus, in this paper we approach TIS recognition from this angle, and apply the different methods that have been developed to deal with imbalanced datasets. The proposed approach has two advantages. Firstly, it improves the results using standard classification methods. Secondly, it broadens the set of classification algorithms that can be used, as some of the class-imbalance methods, such as undersampling, are also useful as methods for scaling up data mining algorithms as they reduce the size of the dataset. In this way, classifiers that cannot be applied to the whole dataset, due to long training time or large memory requirements, can be used when undersampling method is applied. Results show an advantage of class imbalance methods with respect to the same methods applied without considering the class imbalance nature of the problem. The applied methods are also able to improve the results obtained with the best method in the literature, which is based on looking for the next in-frame stop codon from the putative TIS that must be predicted.

Class imbalance methods for translation initiation site recognition

Translation initiation sites (TIS) recognition is one of the first steps in gene structure prediction, and one of the common components in any gene recognition system. Many methods have been described in the literature to identify TIS in transcripts such as mRNA, EST and cDNA sequences. However, the recognition of TIS in DNA sequences is a far more challenging task, and the methods described so far for transcripts achieve poor results in DNA sequences. Most methods approach this problem taking into account its biological features. In this work we try a different view, considering this classification problem from a purely machine learning perspective. From the point of view of machine learning, TIS recognition is a class imbalance problem. Thus, in this paper we approach TIS recognition from this angle, and apply the different methods that have been developed to deal with imbalance datasets. Results show an advantage of class imbalance methods with respect to the same methods applied without considering the class imbalance nature of the problem. The applied methods are also able to improve the results obtained with the best method in the literature, which is based on looking for the next in-frame stop codon from the putative TIS that must be predicted.

The APSES Transcription Factor Vst1 is a Key Regulator of Development in Microsclerotium and Resting Mycelium Producing Verticillium Species

Plant pathogens of the genus Verticillium pose a threat to many important crops worldwide. They are soil-borne fungi which invade the plant systemically, causing wilt symptoms. We functionally characterized the APSES family transcription factor Vst1 in two Verticillium species, V. dahliae and V. nonalfalfae, which produce microsclerotia and melanized hyphae as resistant structures, respectively. We found that, in V. dahliae Δvst1 strains, microsclerotium biogenesis stalled after an initial swelling of hyphal cells and cultures were never pigmented. In V. nonalfalfae Δvst1, melanized hyphae were also absent. These results suggest that Vst1 controls melanin biosynthesis independent of its role in morphogenesis. The absence of vst1 also had a great impact on sporulation in both species, affecting the generation of the characteristic verticillate conidiophore structure and sporulation rates in liquid medium. In contrast with these key roles in development, Vst1 activity was dispensable for virulence. We performed a microarray analysis comparing global transcription patterns of wild-type and Δvst1 in V. dahliae. G-protein/cyclic adenosine monophosphate (G-protein/cAMP) signalling and mitogen-activated protein kinase (MAPK) cascades are known to regulate fungal morphogenesis and virulence. The microarray analysis revealed a negative interaction of Vst1 with G-protein/cAMP signalling and a positive interaction with MAPK signalling. This analysis also identified Rho signalling as a potential regulator of morphogenesis in V. dahliae, positively interacting with Vst1. Furthermore, it exposed the association of secondary metabolism and development in this species, identifying Vst1 as a potential co-regulator of both processes. Characterization of the putative Vst1 targets identified in this study will aid in the dissection of specific aspects of development.

The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum

We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium albo-atrum. We determined that its larger segment (dsRNA1) was 1747 base pairs in length and potentially encoded an RNA-dependent RNA polymerase of 539 amino acids, whereas the smaller segment (dsRNA2) was 1517 base pairs long and was predicted to encode a capsid protein of 435 amino acids. Homology searches and phylogenetic analysis confirmed that, as expected from its dsRNA banding profile, the identified virus was a new member of the family Partitiviridae, and we have therefore designated it V. albo-atrumpartitivirus 1 (VaaPV1). This is the first report of a mycovirus identified in V. albo-atrum.