2.1. E. necator Developmental Stages
To investigate the developmental time-course of the pathogen in Chinese wild V. quinquangularis clone “Shang-24” and V. pseudoreticulata clone “Hunan-1”, we conducted a microscopy study of conidiospore germination and hyphal development during a 5-day time period. Microscopic images of 24, 48 and 120 hpi are presented in .
Figure 1 Progression of PM on V. quinquangularis clone “Shang-24” and V. pseudoreticulata clone “Hunan-1” leaves. Shown are representative images taken at 24, 48, and 120 hpi with conidiospores. Spores and hyphae were stained with (more ...)
Histological observations revealed that conidial germination, germ tube formation, and development of the appressorium occurred on leaves of both grapevine clones (). At 24 hpi, conidiospores produced germ tube and appressoria on both V. quinquangularis and V. pseudoreticulata leaves. In V. quinquangularis leaves, most epidermal cells invaded by the conidiospores led to a hypersensitive response and the attachment by the conidiospores induced H2O2 accumulation, as indicated by brown staining due to DAB polymerization (). At this stage, the staining was obvious but faint and it appeared to be in the wall of mesophyll cells where they made contact with the attacked epidermal cells. Mesophyll that had no contact with the attacked epidermal cells did not show any brown staining, and the staining was intense on reaching 120 hpi in V. quinquangularis leaves. In contrast, in V. pseudoreticulata leaves during the early phase of infection, no DAB staining appeared, and even at 120 hpi only very weak DAB staining was detected in mesophyll cells at few infection sites. The further infection led to the formation of colonies with dense secondary hyphae on V. pseudoreticulata leaves, but only small colonies with sparse hyphae on V. quinquangularis leaves at 120 hpi (). On the basis that the first infection occurred within 24 hpi in Chinese wild V. quinquangularis “Shang-24” (), in the present study we chose leaves at 12 to 120 hpi for the SSH library construction.
2.2. SSH Library Construction, EST Sequencing, Assembly, and Annotation
To identify genes that are potentially involved in V. quinquangularis
clone “Shang-24” resistance to E. necator
, an SSH library was constructed with mock-inoculated “Shang-24” leaves as the driver and E. necator
-inoculated leaves as the tester. A total of 605 clones from the SSH library were selected and PCR analysis of white E. coli
colonies containing cDNA inserts in the pGEM-T Easy vector showed that the size of the cDNA inserts ranged from 150 to 1000 bp. This confirmed the quality of the subtracted cDNA library. A total of 526 clones were identified to contain inserts larger than 200 bp, and were selected for sequencing. After removing low quality sequences and sequences of bacterial origin, 492 high quality ESTs ranging between 200 and 1000 bp were obtained. These ESTs were further assembled into 266 unigenes, among which 101 were contigs and 165 were singletons (Table S1
). The number of EST members in unigenes varied from one to 20 (), with the majority of unigenes present in low copy numbers.
Distribution of number of EST members in each grape unigene.
The “Shang-24” unigenes were further analyzed by searching against the NCBI (National Center for Biotechnology Information) nr database using the BLASTX program. A total of 205 (77.1%) unigenes including 77 contigs and 128 singletons showed significant similarities to known functional gene sequences in the database, 25 had matches against genes of unknown function or hypothetic proteins. The remaining 36 sequences (15 contigs and 21 singletons) did not match to any known gene sequences in the database.
The unigenes from E. necator-inoculated grapevine leaves were further assigned gene ontology (GO) terms. Based on the GO annotations, unigenes were classified into high-level plant specific GO slims within the two ontology categories, namely, molecular function and biological process. A total of 176 unigenes were assigned to 22 GO slims within the molecular function category and 188 unigenes to 41 GO slims within the biological process category (). Within the molecular function category, catalytic activity and binding were the most abundant GO slims (). Response to stress was the most abundant GO slim within the biological process category, which contained responses to stress, abiotic stimulus, and chemical stimulus, which is consistent with the fact that the unigenes were derived from tissues subjected to E. necator inoculation in Chinese wild V. quinquangularis ().
Functional classification of V. quinquangularis “Shang-24” unigenes within (A) molecular function categories and (B) biological process categories, respectively.
2.3. PM Resistance-Related Genes
Although a large amount of E. necator-induced sequence information was generated from Chinese wild V. quinquangularis in this study, only 87 unigenes that were thought to be more relevant to PM resistance because of their defense-related roles and the higher number of those genes were sorted into different categories, based on their putative functions (). The largest category, consisting of 22 (8.3%) unigenes, was predicted to encode resistance and stress-related proteins, such as PR protein, senescence-associated protein, glutathione S-transferase 3, F-box family protein, chitinase, proline-rich protein, and catalase. Nineteen (7.1%) unigenes were related to metabolism, 15 (5.6%) encoding proteins and enzymes putatively involved in photosynthesis and energy, the majority of which encoded ribulose-1, 5-bisphosphate carboxylase/oxygenase activase, 7 (2.6%) unigenes were related to protein synthesis and fate, and genes involved in signal transduction, transport, and transcription accounted for 3.0%, 4.5%, and 1.5%, respectively.
PM-resistance related genes isolated from the SSH library in V. quinquangularis.
2.4. Expression Analysis of E. necator-Responsive Genes by qRT-PCR
To further confirm the expression patterns of identified genes during the interaction between grapevine and E. necator at different time points, qRT-PCR was performed with 11 genes represented by one EST and two genes by two or more ESTs on E. necator- and mock-inoculated samples at eight time points (0, 6, 12, 24, 48, 72, 96, and 120 hpi). These 13 genes in different functional groups were selected for the qRT-PCR analysis because of defense-related roles of their homologs in hypersensitivity, ubiquitin/proteasome pathway and phenylpropanoid synthesis reported previously, and because of their novel sequences. Nine of them corresponded to three functional categories: metabolism (3 genes), protein synthesis and fate (3 genes) and response to defense (3 genes) (), and four had no match to any sequences in the GenBank nr database. Distinct expression patterns of these genes were observed in resistant and susceptible grapevines in response to E. necator and most of the selected genes were strongly elevated by E. necator only in the resistant material “Shang-24”, while not in the susceptible material “Hunan-1” ().
Figure 4 Real-time quantitative RT-PCR analysis of transcript accumulation in two grapevine genotypes in response to E. necator at 0, 6, 12, 24, 48, 72, 96, and 120 hpi. Data represents means of triplicate data. Expression profiles of genes in the disease/defense (more ...)
Three genes involved in defense (UN073, UN116, and UN174), which encode a thaumatin-like protein, a senescence-associated protein, and a class IV chitinase, respectively, shared similar expression patterns in the resistant plant. They did not start increasing until 12 hpi, and all of them reached their maximum expression levels at 24 hpi, then decreased gradually from 48 to 120 hpi; while in the susceptible material, the expression levels of these three genes were consistently close to the control levels throughout the entire time course ().
Three genes in the protein synthesis and fate group (UN134, UN135, and UN203), coding for a coronatine insensitive 1 (COI1), an F-box family protein, and a jasmonate ZIM-domain protein, respectively, were activated strongly by E. necator from 6 to 72 hpi in the resistant material “Shang-24”, while in “Hunan-1”, their transcripts consistently remained at control levels throughout the entire time course ().
Three genes in the category of metabolism (UN044, UN182, and UN260) showed homology to S-adenosylmethionine synthetase, chalcone-flavanone isomerase, and phenylalanine ammonia-lyase, respectively (). They all sharply increased as early as at 6 hpi in “Shang-24” and reached the maximal levels at 48 hpi, whereas their expression did not change significantly in “Hunan-1” upon E. necator-inoculation ().
The expression pattern of four novel genes that had no hits in the database at different time points post inoculation of E. necator is shown in . Two genes (UN113 and UN235) were induced as early as 6 hpi in “Shang-24”, but remained at control levels (UN113) or did not increase until 24 hpi (UN235) in “Hunan-1”. Another gene (UN168) increased at 6 hpi and reached the maximum expression level at 24 hpi in “Shang-24” in response to E. necator, while in “Hunan-1”, the reaction was delayed until 72 hpi. Gene UN103 showed a completely different expression pattern with other genes in our experiment: induced at 6 hpi and reached its maximum expression level at 12 hpi in “Hunan-1” though also slightly increased in “Shang-24” ().