Summary: During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) to visualize proteins secreted during C. fulvum–tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate‐binding modules. Ecp7 encodes a small, cysteine‐rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)‐mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology‐based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.

Summary: A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus‐induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non‐host bacterial pathogens. It also suppressed the HR triggered by transient co‐expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf‐4, indicating that plant PCD can be independent of cathepsin B. The non‐host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.

The molecular signals and pathways that govern biotic and abiotic stress responses in sugarcane are poorly understood. Here we describe SodERF3, a sugarcane (Saccharum officinarum L. cv Ja60-5) cDNA that encodes a 201-amino acid DNA-binding protein that acts as a transcriptional regulator of the ethylene responsive factor (ERF) superfamily. Like other ERF transcription factors, the SodERF3 protein binds to the GCC box, and its deduced amino acid sequence contains an N-terminal putative nuclear localization signal (NLS). In addition, a C-terminal short hydrophobic region that is highly homologous to an ERF-associated amphiphilic repression-like motif, typical for class II ERFs, was found. Northern and Western blot analysis showed that SodERF3 is induced by ethylene. In addition, SodERF3 is induced by ABA, salt stress and wounding. Greenhouse-grown transgenic tobacco plants (Nicotiana tabacum L. cv. SR1) expressing SodERF3 were found to display increased tolerance to drought and osmotic stress.

Summary: Plant defensins are small cysteine‐rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani, Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae. Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to R. solani in greenhouse conditions and, furthermore, they are protected against P.hyoscyami f.sp. tabacina and P. nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.