Gene differential expression of Phytophthora sojae during sporangium formation process

The gene differential expression of Phytophthora sojae during early stage (0 to 7 h) of sporangium formation was studied by differential display reverse transcription polymerase chain reaction (DDRTPCR) in the present study. Thirty out of 78 primer combinations were selected and 90 stable differential bands were obtained. Thirty-nine positive differential fragments were found in the experiments by using the reverse northern blot, in which 36 sequences of differential fragments were obtained after sequencing. Homology search and analysis were tested by BLASTX in the P. sojae genome database. The results showed that 23 fragments had significant similarities with proteins in P. sojae genome database, 15 of which matched with the known functional proteins involved in metabolism, cellular processes, and signal transduction processes. Data mining and bioinformatics analysis on 4 fragments which redundancy was greater than 5 showed that sequence AR15-258 had a very high expression level in Phytophthora mycelium, which may be related to mycelium infection or hunger; AR10-263 and GR1304 were only found in Phytophthora, which might be unique expression in this genus. The analysis and research on important regulatory genes controlling sporangium formation of P. sojae was a foundation for studying of key genes controlling growth and development of P. sojae.


INTRODUCTION
Phytophthora root rot of soybean, caused by infection of Phytophthora sojae, is a worldwide recognized soybean disease due to its large infection area and severity (Tyler, 2007).It has also been listed as one of the destructive diseases of soybean (Tyler et al., 2006).It is basically a kind of single cycle soil-borne disease; therefore, the infection mainly depends on whether the oospores survived in the soil could germinate to form sporangia and whether the zoospores could be released from sporangia or not.With the understanding and knowledge of molecular mechanism of these key aspects, we can control the production of inoculums and thus control the infection and harm fundamentally.*Corresponding author.E-mail: jzhwen2000@yahoo.com.cn.
Previous studies indicated that two genes are important in formation of sporangium of Phytophthora infestans, one of which is Pigpb1, which codes G-protein β-subunit, Pigpb1-silenced mutants form very few sporangia (Latijnhouwers and Govers, 2003).Another is piCdc14, which expresses in sporangiophore initials, coding Cdc14 proteins to regulate mitosis and the cell cycle (Ah-Fong and Judelson, 2003).In addition, Blanco and Judelson (2005) identified an interactor of the zoosporogenesisinduced kinase Pipkz1.Pibzp1 is necessary for normal zoospore movement, essential for appressoria formation and plant infection.PsGPR11-silenced transformants in P. sojae exhibited no differences in sporangium production or size; however, the release of zoospores from sporangia was severely impaired, and about 50% of the sporangia did not completely release their zoospores (Wang et al., 2010).So far, no results on genes relating to sporangium formation of P. sojae have been reported.The recent completion of P. sojae genome sequencing and the development of effective molecular genetic tools offer new opportunities for examining the genetic basis of P. sojae biology, physiology and pathogenicity (Tyler et al., 2006;Lamour et al., 2007).
Differential display reverse transcription PCR (DDRT-PCR) is an effective method on screening of differentially expressed genes from organism's cells and tissues (Liang and Arthur, 1992).This method has also been widely applied in the study of differentially expressed genes of fungi (Takemoto et al., 2000;Nagee et al., 2008).The purpose of this research was to study gene differential expression in formation (0 to 7 h) of P. sojae sporangium with DDRT-PCR technique, analyze important regulatory genes controlling sporangium formation, in order to lay a foundation for study of the key genes relating to growth and development of P. sojae.

Strain of P. sojae and induction of sporangia
P. sojae Ps-597-3 with a pathotype of 1a3c7 isolated from soybean plant infected with Phytophthora root rot disease was provided by the Plant Pathology laboratory of Northeast Agricultural University (China).
Pure vegetative mycelia of Ps-597-3 were produced by the method of inoculating it on a layer of sterile cellophane on surface of carrot agar medium and cultivated for several days under 25°C.A mycelia disc was taken with a sterile hole puncher and placed in a sterile Petri-dish, then added with sterile distilled water to induce the formation of sporangia under 25°C for 6 days, changing the water every 3 h.The mycelia disc was taken respectively at 0, 0.5, 1, 3, 4, 6 and 7 h during the process of induction and quickly dried with sterile filter paper.The samples were preserved at -80°C after frozen in liquid nitrogen for extraction of total RNA.

Polyacrylamide gel electrophoresis and achievement of differential bands
In brief, 5 μL of PCR product added with 2 μL of loading buffer was denaturated and placed on ice before 6% polyacrylamide gel electrophoresis.Differential bands produced by 7 samples amplified with the same pair of primer were directly cut from the gel after silver staining and recovered by boiling.Then, 5 μL of supernatant was taken as template for secondary amplification with the same PCR system and procedures as the aforementioned differential PCR amplification.PCR amplification products were run by agarose gel electrophoresis and bands of the similar size as the original band were purified and recovered with UNIQ-10 column DNA gel extraction kit (Sangon, Shanghai, China).

Differential fragments test with reverse northern blot
Extraction, purification of total RNA of P. sojae mycelia and reverse transcription synthesis of cDNA were repeated and the purified cDNA was labeled with Digoxigenin (Roch) and hybridized with the purified recovered differential fragments and chromatography.The specific method was referred to instructions of DIG High Prime DNA Labeling and Detection Starter Kit II (TaKaRa, Dalian, China).

Cloning and sequencing of differential fragments and sequence analysis
Positive differential fragments after hybridization was connected with pGM-T vector (Beijing TIANGEN, Beijing, China) and cultivated in SOC medium containing IPTG, X-gal and ampicillin at 37°C after competent cells of Escherichia coli DH5α hot-striked with DNA recombinant plasmid.White spot was inoculated to LB liquid culture medium at 37°C overnight and PCR reaction was done with 1 µL liquid according to the procedures of differential PCR amplification.Positive clones from electrophoresis test were sent to Harbin Boshi Biotech Co., Ltd. for sequencing.
NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi)BLASTX and the P. sojae Genome Database release version 1.1 (http://genome.jgipsf.org/sojae1/sojae1.home.html)BLASTX were used for homology sequence analysis of the results and the results were only considered as important when Expectation values <1e-4 (Trudy et al., 2007).Both E value <1e-4 and the BLOSUM62 scoring matrix were used in Blast Algorithms.Finally, fragments with a redundancy greater than 5 were used for data mining and bioinformatics analysis (Chen et al., 2007).

Screening of primer combinations
In the present study, three kinds of anchor primers and 26 differential displayed random primers, which were 78 primer combinations in total, were used for DDRT-PCR amplification of 21 cDNAs.Among them, 30 primer combinations with anchor primer P A , P C , P G and random primer including R 1 , R 2 , R 4 , R 7 , R 10 , R 15 , R 19 , R 22 , R 24 and R 26 had clear-band of PCR product.The number of bands amplified with different primer combinations was varied, and the size of DNA fragments amplified ranged from 100 to 750 bp.Amplification results of primers P A R 17 and P C R 10 are shown in Figure 1.

Differential display PCR amplification
The PCR products from the 30 primer combinations were tested with 6% denatured polyacrylamide gel electrophoresis (Figure 2).In total, 101 differential bands ranging from 100 to 400 bp were amplified and could be divided into four categories: (1) fragments (band a) that appeared in the control group, but not shown in the induction group; (2) fragments (band b) that appeared in the control group, but gradually disappeared in the induction group; (3) fragments (band c) that did not appear in the control group, but shown instantly in the induction group; (4) fragments (band d) that did not appear in the control group, but shown in the induction group in a certain time and expressed from beginning to end.
These 101 differential fragments were cut for another PCR amplification and purification, and 90 stable differential bands were obtained, in which 24 differential bands contained anchor primer P A , 45 contained anchor primer P G , and 21 contained anchor primer P C .

Reverse northern blot
Reverse northern blot was applied in this study to identify positive differential bands.Digoxigenin (Roch) was used to label cDNA synthesized from reverse transcription, which was used as a probe hybrid with differential bands on nylon membrane for a secondary amplification and screened for the real differential fragments.The results of reverse northern blot showed that 13 out of 24 differential fragments with anchor primer P A , 18 out of 45 with anchor primer P G and 8 out of 21 with anchor P C were positive.Thus, in total, there were 39 positive differential fragments, and the remaining 51 differential fragments were false positive and false-positive rate was 56.7% (Figure 3).

Sequencing and analysis of differential fragment
Thirty-nine differential fragments were cloned, verified, and delivered to Harbin Boshi Biotech Co., Ltd., and a total of 36 differential fragments were sequenced.Fragments were named as anchor primers (A / C / G) + random primers (R 1 to R 26 ) + (-) + fragment length, such as AR 1 -163.
Sequences of 36 differential fragments were compared in the P. sojae Genome Database with BLASTX (Table 1).The results suggested that 23 differential fragments significantly matched the proteins in genome database of P. sojae, and the matching rate was 63.9%, in which 15 matches were known functional proteins involved in metabolism, cellular processes and signal transduction process; 13 proteins had no significant match; four fragments, which redundancies were greater than 5, were respectively GR 1 -304, GR 1 -373, AR 10 -263 and AR 15 -258 (mRNA differential display pattern was shown in Figure 4), and the function of comparison proteins in genome database of P. sojae were respectively cytokinesis regulating protein-like, diacylglycerol acyltransferase (DAGAT), cellulose-binding elicitor and lectin-like(CBEL) protein and putative ABC transporter protein.
Data mining and bioinformatics analysis of 4 fragments with a redundancy greater than 5 (Table 2) showed that 26 EST matched with AR 15 -258 in Phytophthora mycelium database, AR 15 -258 also matched with one EST in P. sojae infection database and hunger database of Phytophthora, respectively.The results indicated that AR 15 -258 was highly expressed in Phytophthora mycelium, which could be possibly related to mycelium infection or hunger; no EST matched with AR 10 -263 was found in Phytophthora database, which indicated that AR 10 -263 was poorly expressed in Phytophthora.Homologous genes were found in GR 1 -304, GR 1 -373 and AR 15 -258 as in P. ramorum and P. capsici in 4 fragments with redundancy greater than 5, no homologous genes were found in AR 10 -263 as in P. ramorum and P. capsici; at the mean time, AR 10 -263 and GR 1 -304, which were unique expressions, were only found in Phytophthora.

Material selection
One of the characteristics of DDRT-PCR technique is high sensitivity, which means a smaller difference on genetic background of the test material leads to lower false positive rate.Differential expressed genes induced by sterile water in formation of P. sojae sporangium were studied in this experiment.Pure vegetative mycelia free from sporangium and oospore were required in order to reduce the differences between test materials.Cellophane has been applied on the fungal culture before, including Phytophthora parasitica (Gaulin et al., 2002) and Magnaporthe grisea (Sun et al., 2006;Mehrabi et al., 2008).However, those applications were only for an easier observation of the infection and movement of the fungus.Carrot agar medium with a layer of sterile cellophane was used in this experiment for cultivation of P. sojae.The mycelium grew slower on the cellophane than directly on carrot agar medium; however, oospores and sporangia were not found in 10 days, which means difference among material was reduced with no affection from oospores and sporangia.Meanwhile, medium would not be attached to mycelium either, which was better for RNA extraction.The sterile water was used in this research for sporangium induction, as the sporangium of P. sojae could not be induced on solid medium.
Researches on growth and development of P. sojae are still inadequate although the economic losses caused by this pathogen were well focused.The purpose of this test was to study genes associated with formation of P. sojae sporangium.Samples were continuously taken at early stage of sporangium formation to extract total RNA for differential display reverse transcription polymerase chain reaction (DDRT-PCR) test, in order to find the key genes regulating the formation of sporangium.Mycelia at 0 h, instead of non-induced mycelia at the same stage, were used as control in screening differential fragments; this not only saved time, but also allowed comparison of mycelia induced by sterile water at various stages.

Function analysis of differential fragments
Cellulose binding elicitor lectin (CBEL) can recognize cellulose; it is related with natural cell wall sedimentation of Phytophthora and can thicken the mycelium (Gaulin et al., 2002).Differential fragments AR 10 -263 and CBEL protein (Mateos et al., 1997) have important similarities, which included expression at 0 to 3 h in P. sojae mycelium, causing cell wall of P. sojae mycelium settled normally, could lead to cell wall thickening and thus provides condition for formation of sporangium.
Diacylglycerol acyltransferase (DAGAT) was strongly induced at the beginning of fat synthesis (Shockey et al., 2006); triacylglycerol was catalyzed into glycerol and fatty acyl-S-CoA (Zou et al., 1999), which regulates the content of triglyceride in seed development (Jako et al., 2001).Triacylglycerol were stored as energy in the rapidly growing cells, spores and various regenerating structures in fungi.Differential fragment GR 1 -373, which was highly expressed at 4 h in P. sojae mycelium induced by sterile water shared essential similarity with DAGAT.It could also be involved in triacylglycerol synthesis and provide energy during tip growth of P. sojae mycelium and sporangium formation.
In addition, differential fragment GR 1 -304 sharing great similarity with cytokines is regulatory protein (Sasaki et al., 2002), which showed high expression in P. sojae mycelium at 4 h induced by sterile water.It could be related to cytokinesis regulation of P. sojae, promoting the formation of zoospores by protoplasm dissever of enlarged cytoplasm inside the hypha.
PDR5 located in plasma membrane of the yeast can evacuate a large variety of anti-cancer drugs from cells (Sasaki et al., 2002).ABC transporter protein, similar to PDR5 and coded by TUR2, could possibly eliminate toxic metabolites out of cells under stress conditions Smart and Fleeting, 1996).Differential fragment AR 15 -258 and assumed ABC (ATP-binding cassette) carrier PDR protein have an important similarity.It was highly (expressed in P. sojae mycelium at 7 h induced by sterile water, which was probably due to lack of nutrition when P. sojae cells were under water stress and thus resulted in certain toxic metabolites requiring discharge by ABC transporter protein.
In summary, when P. sojae mycelia was induced by sterile water, CBEL gene was first regulated and 3 h later CBEL silencing led to mycelia cell wall thickening, which provided material basis for sporangium formation; and DAGAT gene was regulated to synthesize triacylglycerol and to provide energy for development of hypha tip of P. sojae as well as formation of sporangium.At the same time, cytokinesis regulatory proteins were controlled to promote the formation of zoospores by protoplasm dissever of enlarged cytoplasm inside the hypha and enhance sporangium formation; and some toxic metabolites were excreted in the process of sporangium formation.The aforementioned was only a speculation of function of each cDNA clone sequence of P. sojae sporangium formation induced by sterile water and a large number of tests were still to be done as proof.The functions of many differential fragments were still unknown in this study.Complete sequenced genes are essential to discover function and regulation mechanism of differential expressed genes induced by sterile water in formation of P. sojae sporangium and much further studies are needed.

Figure 1 .
Figure 1.Electrophoresis profiles of the PCR products amplified by primer combination PAR17 (A) and PCR10 (B) from the single strand cDNA of P. sojae mycelia induced with sterile water.M: Marker DL2000; A: Combination of primer PA and R17; B: Combination of primer PC and R10; lanes 1 to 7: PCR amplification products from the single strand cDNA of P. sojae mycelia at 0, 0.5, 1, 3, 4, 6 and 7 h after induced with sterile water.

Figure 2 .
Figure 2. Partial mRNA differential display profiles of P. sojae during sporangium formation process.M: Marker DL2000; A: Combination of primer PA and R1; B: Combination of primer PG and R19; lane 1 to 7: PCR amplification products from single strand cDNA of P. sojae mycelia at 0, 0.5, 1, 3, 4, 6 and 7 h after induced with sterile water.Differential fragments are pointed by arrows.

Figure 3 .
Figure 3. Reverse northern blot for the differential fragments.A: 24 differential fragments with anchor primer PA; B: 45 with anchor primer PG; C: 21 with anchor primer PC.

Table 1 .
Results of BLASTX comparison of 36 differential expressed sequences in genome database (version1.1) of P. sojae during the early stage (0 to 7 h) of sporangium formation under sterile water stress.

Table 2 .
Data mining and bioinformatics analysis of differential fragments (redundancy >5).